De Novo Design of Potent and Selective Interleukin Mimetics

ABSTRACT

De novo designed polypeptides that bind to IL-2 receptor βν c  heterodimer (IL-2Rβν c ), IL-4 receptor αν c  heterodimer (IL-4Rαν c ), or IL-13 receptor α subunit (IL-13Rα) are disclosed, as are methods for using and designing the polypeptides.

CROSS REFERENCE

This application is a Continuation of U.S. patent application Ser. No.16/572,038, filed Sep. 16, 2019, which is a Continuation ofInternational Application No. PCT/US2019/038703, filed Jun. 24, 2019,which claims priority to U.S. Provisional Application No. 62/768,733,filed Nov. 16, 2018, and U.S. Provisional Application No. 62/689,769,filed Jun. 25, 2018, the disclosures of which are hereby incorporated byreference in their entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with Government support under contract AI051321awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND

The considerable potential of central immune cytokine interleukins suchas IL-2 and IL-4 for cancer treatment has sparked numerous efforts toimprove their therapeutic properties by mutation and/or chemicalmodification. However, because these approaches are closely tied tonative IL-2 or IL-4, they cannot eliminate undesirable properties suchas low stability and binding to the IL-2 receptor α subunit (IL-2Rα), toIL-4 receptor αν_(c) heterodimer (IL-4Rαν_(c)), or to IL-13 receptor αsubunit (IL-13Rα).

SUMMARY

In one aspect, a method is provided. A computing device determines astructure for a plurality of residues of a protein where the structureof the plurality of residues provides a particular receptor bindinginterface. The computing device determines a plurality of designedresidues using a mimetic design protocol provided by the computingdevice, wherein the plurality of designed residues provide theparticular receptor binding interface, and wherein the plurality ofdesigned residues differ from the plurality of residues.

The computing device determines one or more connecting helix structuresthat connect the plurality of designed residues. The computing devicedetermines a first protein backbone for the protein by assembling theone or more connecting helix structures and the plurality of designedresidues over a plurality of combinations. The computing device designsa second protein backbone for the protein for flexibility and low energystructures based on the first protein backbone. The computing devicegenerates an output related to at least the second protein backbone.

Also included are non-naturally occurring proteins prepared by themethods described herein. The non-naturally occurring proteins can becytokines, for example, non-naturally occurring IL-2 or IL-4 (alsoreferred to herein as IL-2, IL-2/15 mimetics or IL-4 mimetics).

In another aspect, a computing device is provided. The computing deviceincludes one or more processors; and data storage that is configured tostore at least computer-readable instructions that, when executed by theone or more processors, cause the computing device to perform functions.The functions include: determining a structure for a plurality ofresidues of a protein that provides a particular receptor bindinginterface; determining a plurality of designed residues using a mimeticdesign protocol, wherein the plurality of designed residues provide theparticular receptor binding interface, and wherein the plurality ofdesigned residues differ from the plurality of residues; determining oneor more connecting helix structures that connect the plurality ofdesigned residues; determining a first protein backbone for the proteinby assembling the one or more connecting helix structures and theplurality of designed residues over a plurality of combinations;designing a second protein backbone for the protein for flexibility andlow energy structures based on the first protein backbone; andgenerating an output related to at least the second protein backbone forthe protein.

In another aspect, a non-transitory computer-readable medium isprovided. The non-transitory computer-readable medium is configured tostore at least computer-readable instructions that, when executed by oneor more processors of a computing device, cause the computing device toperform functions. The functions include: determining a structure for aplurality of residues of a protein that provides a particular receptorbinding interface; determining a plurality of designed residues using amimetic design protocol, wherein the plurality of designed residuesprovide the particular receptor binding interface, and wherein theplurality of designed residues differ from the plurality of residues;determining one or more connecting helix structures that connect theplurality of designed residues; determining a first protein backbone forthe protein by assembling the one or more connecting helix structuresand the plurality of designed residues over a plurality of combinations;designing a second protein backbone for the protein for flexibility andlow energy structures based on the first protein backbone; andgenerating an output related to at least the second protein backbone forthe protein.

In another aspect, a device is provided. The device includes: means fordetermining a structure for a plurality of residues of a protein thatprovides a particular receptor binding interface; means for determininga plurality of designed residues using a mimetic design protocol,wherein the plurality of designed residues provide the particularreceptor binding interface, and wherein the plurality of designedresidues differ from the plurality of residues; means for determiningone or more connecting helix structures that connect the plurality ofdesigned residues; means for determining a first protein backbone forthe protein by assembling the one or more connecting helix structuresand the plurality of designed residues over a plurality of combinations;means for designing a second protein backbone for the protein forflexibility and low energy structures based on the first proteinbackbone; and means for generating an output related to at least thesecond protein backbone for the protein. In another aspect,non-naturally occurring polypeptides are provided comprising domains X1,X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence at least 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to EHALYDAL (SEQ ID NO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence at least 25%%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to YAFNFELI (SEQ ID NO:2);

(d) X4 is a peptide comprising the amino acid sequence at least 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to ITILQSWIF (SEQ ID NO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and

wherein the polypeptide binds to IL-2 receptor βν_(c) heterodimer(IL-2Rβν_(c)), IL-4 receptor αν_(c) heterodimer (IL-4Rαν_(c)), or IL-13receptor α subunit (IL-13Rα).

In other aspects are provided pharmaceutical compositions comprising oneor more polypeptide disclosed herein and a pharmaceutically acceptablecarrier, recombinant nucleic acids encoding a polypeptide disclosedherein, expression vectors comprising the recombinant nucleic acidsdisclosed herein, and recombinant host cells comprising one or moreexpression vector disclosed herein. In a further aspect, methods fortreating cancer are provided, comprising administering to a subjecthaving cancer one or more polypeptide, recombinant nucleic acid,expression vector comprising the recombinant nucleic acid, and/orrecombinant host cells disclosed herein or a pharmaceutical compositionthereof in an amount effective to treat the tumor.

DESCRIPTION OF THE DRAWINGS

The following figures are in accordance with example embodiments:

FIG. 1A-1D. Computational design of de novo cytokine mimetics. FIG. 1A)The designed mimetics have four helices; three mimetic IL-2 interactionswith hIL-2Rβν_(c), while the fourth holds the first three in place. Top:in the first generation of designs, each of the core elements of IL-2(helices H1-H4) were independently idealized using fragment-assemblyfrom a clustered ideal fragment database (size: 4 a.a.); bottom: in thesecond generation of designs the core elements were instead built usingparametric equations that recapitulate the shape of each disembodiedhelix, allowing changes in the length of each helix by +/−8 a.a.; FIG.1B) Pairs of helices were reconnected using ideal loop fragments (size:4 a.a. or 7 a.a., for gen-1 and gen-2 respectively, see Methods),representative examples are shown with newly built elements connectingeach pair of helices; FIG. 1C) The helix hairpins generated in FIG. 1Bwere assembled in all possible combinations to generate fully connectedprotein backbones; FIG. 1D) The designs and experimentally maturedversions were tested for binding by yeast display, and those exhibitinghigh affinity binding were recombinantly expressed (E. coli) and testedfor binding using surface plasmon resonance and IL-2 like phospho-STAT5(pSTAT5) signaling. The results for 3 designs of the first generationand 10 designs from the second generation are shown in the 2D-plot insolid symbols. The open star is Neoleukin-2/15, the arrow originates inits parent (unoptimized) design.

FIG. 2A-2C. Characterization of neoleukin-2/15. FIG. 2A) From top tobottom: In surface plasmon resonance experiments, neoleukin-2/15 doesnot bind human or murine IL-2Rα, but binds both human and murine IL-2Rβwith similar affinity (K_(d)˜11.2 nM and 16.1 nM, for human and micereceptor, respectively). Like natural IL-2, neoleukin-2/15 binds poorlyto the ν_(c) receptor, and exhibits cooperative binding for both humanand murine IL-2Rβν_(c) (K_(d)˜18.8 nM and 38.4 nM, for the human andmice heterodimeric receptor, while the Kd of native hIL-2 and Super-2are ˜193.6 nM and 300.9 nM, see Table E1). FIG. 2B) top: In-vitro pSTAT5signaling studies demonstrate that neoleukin-2/15 elicits IL-2-likesignaling in human cells (EC₅₀), and activates with identical potency(EC₅₀˜73.0 pM and 49.2 pM on CD25+ and CD25− cells, respectively) humanYT-1 NK cells with or without IL-2Rα expression (CD25); bottom:similarly ex vivo experiments in murine CD4+ primary cells demonstratethat neoleukin-2/15 can also elicit potent IL-2 like signaling in murinecells, and is independent of IL-2Rα expression (EC₅₀˜24 pM and 129 pM onCD25+ and CD25− cells, respectively); FIG. 2C) top: binding experiments(OCTET) show that neoleukin-2/15 can be incubated for 2 hours at 80° C.without any noticeable loss of binding, whereas human and murine IL-2quickly lose activity; bottom: an ex vivo experiment on cultured murinesplenocytes that require IL-2 for survival, demonstrates thatneoleukin-2/15 incubated at 95° C. for 1 hour still drives cell survivaleffectively (˜70% relative luminescence, at 10 ng/ml), while mIL2 andSuper-2 are virtually inactive (˜10% and 0.1%, respectively at 10ng/ml).

FIG. 3A-3E. Structure of neoleukin-2/15 (Neo-2/15) and its ternarycomplex with mIL-2Rβν_(c). FIG. 3A) Top: structural alignment ofneoleukin-2/15 (Neo-2/15) chain A with the design model (r.m.s.d. 1.11 Åfor 100 Cα atoms); bottom: detail of interface helices H1, H3 and H4(numbered according to hIL-2, see FIG. 1). The interface side chains areshown in sticks; FIG. 3B) crystallographic structure of the ternarycomplex of Neo-2/15 with mIL-2Rβ and ν_(c) (r.m.s.d 1.27 Å for the 93modeled Cα atoms of Neo-2/15 in the ternary complex); FIG. 3C)structural alignment of monomeric Neo-2/15 (chain A) with Neo-2/15 inthe ternary complex (r.m.s.d 1.71 Å for the 93 modeled Cα atoms in theternary complex). Helix H4 shows an approximately 4.0+shift of helix H4in the ternary-complex structure compared to the monomeric crystalstructure; FIG. 3D) crystallographic structure of: hIL-2 (cartoonrepresentation). The regions that interact with the IL-2Rβ and γ_(c) aredenoted. The loop-rich region from hIL-2 that interacts with IL-2Rα doesnot exist in the de novo mimetic Neo-2/15. FIG. 3E): crystallographicstructure of neoleukin-2/15 from the ternary complex in “b)” (cartoonrepresentation). The regions that interact with the IL-2Rβ and γ_(c) aredenoted. The loop-rich region from hIL-2 that interacts with IL-2Rα doesnot exist in the de novo mimetic Neo-2/15.

FIG. 4A-4G. Immunogenicity, immunostimulatory and therapeutic activityof neoleukin-2/15. FIG. 4A) Dose escalation effect of neoleukin-2/15(Neo-2/15) in naive mice T cells. Naive C57BL/6 mice were treated dailywith neoleukin-2/15 or mIL-2 at the indicated concentrations (n=2-3 pergroup). After 14 days, spleens were harvested and analyzed by flowcytometry using the indicated markers. The bar plot shows that mIL-2enhanced CD4+ Treg expansion in a dose dependent fashion, while Neo-2/15had little or no effect in expansion of Treg cells. Neoleukin-2/15 drovea higher CD8+:Treg ratio compared to mIL-2; FIG. 4B) Effect of Neo-2/15in mice in an airway inflammation model (20 μg/day/mouse, 7 days).Similar to naive mice, Neo-2/15 does not increase the frequency ofantigen-specific CD4+ Foxp3+T_(regs) in the lymphoid organs, and iscomparably effective to mIL-2 in increasing the frequency of lungresident (Thy1.2—by intravascular labeling) CD8+ T cells; FIG. 4C)Neoleukin-2/15 does not have detectable immunogenicity. C57BL/6 micewere inoculated with 5×10⁵ B16F10 cells by subcutaneous injection.Starting on day 1, mice were treated daily with neoleukin-2/15 (10 μg)or equimolar mIL-2 by intraperitoneal (i.p.) injection (n=10 for eachgroup). After 14 days, serum (antiserum) was collected and IgG wasdetected by ELISA in plates coated with fetal bovine serum (FBS 10%,negative control), neoleukin-2/15, mIL-2, hIL-2, or Ovalbumin (OVA) asnegative control (the dotted line shows the average of the negativecontrol). Anti-Neo-2/15 polyclonal antibody was used as positive control(black, n=2) and did not cross react with mIL-2 or h-IL2; FIG. 4D)C57BL/6 mice were immunized with 500 μg KO Neo-2/15 in complete Freund'sadjuvant and boosted on days 7 and 15 with 500 μg KO Neo-2/15 inincomplete Freund's adjuvant. Reactivity against KO Neo-2/15 and nativeNeo-2/15, as well as cross-reactivity with mouse IL-2 were determined byincubation of serum (diluted 1:1,000 in PBS) with plate-bound KONeo-2/15, Neo-2/15 or mouse IL-2 as indicated. Serum binding wasdetected using an anti-mouse secondary antibody conjugated to HRPfollowed by incubation with TMB. Data are reported as optical density at450 nm. Top, naive mouse serum; bottom, immunized mouse serum. FIG.4E-4G) Therapeutic efficacy of Neoleukin-2/15: FIG. 4E) BALB/C mice wereinoculated with CT26 tumors. Starting on day 6, mice were treated dailywith i.p. injection of mIL-2 or neoleukin-2/15 (10 μg), or were leftuntreated (n=5 per group). Tumor growth curves (top, show only data forsurviving mice). Survival curves (bottom). Mice were euthanized whenweight loss exceeded 10% of initial weight or when tumor size reached1,300 mm³. FIG. 4F) C57BL/6 mice were inoculated with B16 tumors as in“a)”. Starting on day 1, mice were treated daily with i.p. injection ofneoleukin-2/15 (10 μg) or equimolar mIL-2 (n=10 per group). Twice-weeklytreatment with TA99 was added on day 3. Mice were euthanized when weightloss exceeded 10% of initial weight or when tumor size reached 2,000mm³. Tumor growth curves (top and bottom left). Survival curve, insetshows average weight change (top right). Quantification of cause ofdeath (bottom right). FIG. 4G) Neo-2/15 elicits a higher CD8+:Treg ratiothan mouse IL-2. C57BL/6 mice were inoculated with B16 tumors andtreated by daily i.p. injection as indicated. Treatment with TA99(bottom plot) was started on day 5 and continued twice-weekly. Tumorswere harvested from mice when they reached 2,000 mm³ and analyzed byflow cytometry. The CD8:Treg cell ratio was calculated by dividing thepercentage CD45⁺ CD3⁺ cells that were CD8⁺ by the percentage that wereCD4⁺ CD25⁺ FoxP3⁺.

FIG. 5A-5D. Therapeutic effect of neoleukin-2/15 on colon cancer. FIG.5A) BALB/C mice were inoculated with CT26 tumors. Starting on day-9 andending on day-14, mice were treated daily with i.p. injection of mIL-2or neoleukin-2/15 at the specified concentrations, or were leftuntreated (n=5 per group). Tumor growth curves (top, show only data forsurviving mice). Survival curves (bottom). Mice were euthanized whenweight loss exceeded 10% of initial weight or when tumor size reached1,300 mm³. FIG. 5B-5D) The bar-plots compare the T cell populations forBALB/C mice (n=3 per group) that were inoculated with CT26 tumors andtreated starting from day-6 with by daily i.p. injection of 10 μg ofNeolukin-2/15 or 10 μg mIL-2 or no-treatment (No Tx). On day-14 thepercentage of Treg cells (CD4⁺ CD45⁺ FoxP3⁺, top graph) and CD8:Tregcell ratio ((CD45⁺ CD3⁺ CD8⁺)/Treg, bottom graph) was assessed in: FIG.5B) tumors, FIG. 5C) neighboring inguinal lymph node (LN), and FIG. 5D)spleen.

FIG. 6A-6D. Therapeutic effect of neoleukin-2/15 on melanoma. FIG.6A-6E) Tumor growth curves (bottom) and survival curves (top) forC57BL/6 mice that were inoculated with B16 tumors and treated with low(1 μg/mice/day, a-b) or high doses of neoleukin-2/15 (10 μg/mice/day,c-d). Starting on day 1, mice (n=5 per group) were treated daily withi.p. injection of FIG. 6A): single agent neoleukin-2/15 at 1 μg/mice orequimolar mIL-2 (n=5 per group), or FIG. 6B): the same treatments incombination with a twice-weekly treatment with TA99 (started on day 5).Mice were euthanized when tumor size reached 2,000 mm³. C57BL/6 micewere inoculated with B16 tumors and treated by daily i.p. injection asindicated. FIG. 6C-6D) Similar to “a-b)”, but starting on day 4, micewere treated daily with i.p. injection of 10 μg/mouse of neoleukin-2/15,or equimolar mIL-2, either alone FIG. 6C) or in combination withtwice-weekly TA99 started on day 4 FIG. 6D). Mice were euthanized whentumor size reached 2,000 mm³. The therapeutic effect of Neoleukin-2/15is dose dependent (higher doses are better) and is potentiated in thepresence of the antibody TA99. The experiments were performed once. Inall the growth curves, data are mean±s.e.m. Results were analysed byone-way ANOVA (95% confidence interval), except for survival curves thatwere assessed using the Mantel-Cox test (95% confidence interval).

FIG. 7A-7C. Reengineering of neoleukin-2/15 into a human interleukin-4(hIL-4) mimetic (neoleukin-4). FIG. 7A) Neo-2/15 structurally alignedinto the structure of IL-4 in complex with IL-4Rα and ν_(c) (from PDBcode 3BPL). Fourteen IL-4 residues that contact IL-4Rα and that weregrafted into Neo-2/15 are labeled. FIG. 7B) Neoleukin-4 (Neo-4), a newprotein with sixteen amino acid mutations compared to Neo-2/15. Thesemutations are labeled; thirteen of these were derived from the IL-4residues depicted in panel “a)” that mediate contact with IL-4Rα, andthree of them (H8M, K68I and I98F, underlined in the figure) wereintroduced by directed evolution using random mutagenesis and screeningfor high binding affinity variants. FIG. 7C) Biolayer interferometrydata showing that Neo-4, like IL-4, binds to IL-4Rα alone, has noaffinity for ν_(c) alone, but binds to ν_(c) when IL-4Rα is present insolution.

FIG. 8A-8B. Stimulatory effect of Neoleukin-2/15 on human CAR-T cells.FIG. 8A) Anti-CD3/CD28 stimulated or FIG. 8B) unstimulated human primaryCD4 (top) or CD8 (bottom) T cells were cultured in indicatedconcentrations of human IL2 or neoleukin-2/15. T cell proliferation ismeasured as fold change over T cells cultured without IL2 supplement.Neo-2/15 is as effective as native IL-2 at inducing proliferation ofstimulated CAR-T cells, and more effective than native IL-2 at inducingproliferation of unstimulated CAR-T cells, particularly of unstimulatedCD8 CAR-T cells.

FIG. 9A-9D. Overall sequence conservation in binding residues for eachof the four common helices, combining information from three differentde novo-designed IL-2 mimics. Sequence logos were generated usingcombined data from binding experiments (using the heterodimeric mouseIL-2Rβγc) from three independent SSM mutagenesis libraries forG2_neo2_40_1F_seq27, G2_neo2_40_1F_seq29 and G2_neo2_40_1F_seq36 (FIGS.11-13). All of these proteins are functional high-affinity de novomimetics of mouse and human IL-2, some having topologies that differfrom that of Neo-2/15, but all containing the four Helices H1 (FIG. 9A;Neo-2/15 1-22 is SEQ ID NO:248, IL-2 6-27 is SEQ ID NO:249, IL-15 1-15is SEQ ID NO:250), H3 (FIG. 9B; Neo-2/15 34-55 is SEQ ID NO:251, IL-282-103 is SEQ ID NO:252, IL-15 59-80 is SEQ ID NO:253), H2′ (FIG. 9C;Neo-2/15 58-76 is SEQ ID NO:254, IL-2 50-68 is SEQ ID NO:255, IL-1534-52 is SEQ ID NO:256) and H4 (FIG. 9D; Neo-2/15 80-100 is SEQ IDNO:257, IL-2 111-131 is SEQ ID NO:258, IL-15 93-113 is SEQ ID NO:259).The logos show the combined information for each helix independently.Below each logo, a line graph shows the probability score (higher meansmore conserved) for each amino acid in the Neo-2/15 sequence. The solidhorizontal line highlights positions where the Neo-2/15 amino acid has aprobability score ≥30% (that is, these amino acids contribute moregenerally to receptor binding as they are globally enriched in thebinding populations across all of the de novo IL-2 mimics tested). Thetopology of each helix in Neo-2/15 is shown left of each logo. Thesequences of the Neo-2/15 helices and those of the corresponding helices(structurally aligned) in human IL-2 and IL-15 are shown below thegraphs, highlighting the distinctiveness of the Neo-2/15 helices andbinding interfaces.

FIG. 10A-10D. Experimental optimization of G1_neo2_40. FIG. 10A-10C)Heatmaps for G1_neo2_40 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 10A) 50 nM, FIG. 10B) 2 nM, and FIG. 10C) 0.1 nMIL-2Rβν_(c) heterodimer. Based on these enrichment data, a combinatoriallibrary was designed with nucleotide diversity 1.5×10⁶. FIG. 10D) Aminoacid residues available in the initial combinatorial library aredepicted indicating residues predicted to be advantageous (shown abovethe original sequence) and deleterious (shown below the originalsequence; in the depiction of the original sequence, black indicatesresidues that are represented in the combinatorial library and gray,residues not represented in the combinatorial library.

FIG. 11A-11E. Experimental optimization of G2_neo2_40_1F_seq27. Heatmapsfor G2_neo2_40_1F_seq27 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 11A) 10 nM, FIG. 11B) 1 nM, FIG. 11C) 0.1 nM, andFIG. 11D) 0.1 nM IL-2Rβν_(c) heterodimer. Based on these enrichmentdata, a combinatorial library was designed with nucleotide diversity5.3×10⁶. FIG. 11E) Amino acid residues available in the initialcombinatorial library are depicted indicating residues predicted to beadvantageous; black indicates residues in the starting sequencerepresented in the combinatorial library.

FIG. 12A-12E. Experimental optimization of G2_neo2_40_1F_seq29. Heatmapsfor G2_neo2_40_1F_seq29 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 12A) 10 nM, FIG. 12B) 1 nM, FIG. 12C) 0.1 nM, andFIG. 12D) 0.1 nM IL-2Rβν_(c) heterodimer. Based on these enrichmentdata, a combinatorial library was designed with nucleotide diversity2.9×10⁶. FIG. 12E) Amino acid residues available in the initialcombinatorial library are depicted indicating residues predicted to beadvantageous; black indicates residues in the starting sequencerepresented in the combinatorial library.

FIG. 13A-13E. Experimental optimization of G2_neo2_40_1F_seq36. Heatmapsfor G2_neo2_40_1F_seq36 single-site mutagenesis library showingenrichment at specific positions after consecutive rounds of increasingselection with FIG. 13A) 10 nM, FIG. 13B) 1 nM, FIG. 13C) 0.1 nM, andFIG. 13D) 0.1 nM IL-2Rβν_(c) heterodimer. Based on these enrichmentdata, a combinatorial library was designed with nucleotide diversity2.7×10⁶. FIG. 13E) Amino acid residues available in the initialcombinatorial library are depicted indicating residues predicted to beadvantageous; black indicates residues in the starting sequencerepresented in the combinatorial library.

FIG. 14A-14B. Circular Dichroism (CD) thermal denaturation experimentsfor multiple IL-2/IL-15 de novo designed mimetics, generation-1. FIG.14A) Thermal denaturation curves and FIG. 14B) wavelength scans.

FIG. 15A-15B. Circular Dichroism (CD) thermal denaturation experimentsfor multiple IL-2/IL-15 de novo designed mimetics, generation-1experimentally optimized. FIG. 15A) Thermal denaturation curves and FIG.15B) wavelength scans.

FIG. 16A-16D. Circular dichroism thermal melts for IL-2/IL-15 mimeticdesigns generation-2. FIG. 16A and FIG. 16C) Thermal denaturation curvesand FIG. 16B and FIG. 16D) wavelength scans.

FIG. 17A-17C. Expression, purification, and thermal denaturationcharacterization of neoleukin-2/15. FIG. 17A) SDS Tris-Tricine gelelectrophoresis showing expression and purification over affinitycolumn. FIG. 17B) Circular dichroism at 222 nm during thermal meltingfrom 25° C. to 95° C., showing robust temperature stability. FIG. 17C)Circular dichroism wavelength scans at 25° C., 95° C. and then again 25°C., showing that neoleukin-2/15 does not fully melt at 95° C. andrefolds fully after cooling back to 25° C.

FIG. 18A-18D. Single disulfide stapled variants of neoleukin-2/15 withhigher thermal stability. Structural model of disulfide stabilizedvariants of Neoleukin-2/15 are shown with positions of the mutatedresidues labeled and the disulfide bond shown. Two strategies were usedto generated the disulfide variants: FIG. 18A) internal placement atresidues 38 and 75 and terminal linkage; FIG. 18B) for the terminalvariant, three residues were added to each terminus in order to limitany distortions to the starting structure that would otherwise berequired to form the disulfide bond. CD spectra at 25° C., 95° C. and25° C. after cooling for the internal and terminal disulfide variantsare shown below their structural models. Both variants show very littlesignal loss at 95° C. and complete refolding upon cooling; FIG. 18C)thermal melts of each variant were performed by monitoring CD signal at222.0 nm over a range of temperatures. Each of the disulfide variantsshows improved stability relative the native; FIG. 18D) binding strengthof each variant to IL-2Rβγc was measured by biolayer interferometry.Contrary to disrupting the binding interaction, these data show theintroduction of the disulfide bond improves the binding of the mimeticsto IL-2Rβγc. Both disulfide-bonded variants exhibit an improvement inbinding IL-2Rβγc (Kd˜1.3±0.49 and 1.8±0.26 nM, for the internal andexternal disulfide-staples, respectively, compared to 6.9±0.61 nM forNeo-2/15 under the same experimental conditions), which is consistentwith the expected effect of disulfide-induced stabilization of theprotein's binding site.

FIG. 19A-19B. Robustness of neoleukin-2/15 to single-point cysteinemutants on non-binding interface positions. FIG. 19A) Schematic showingpoint mutant positions in neolukin-2/15 that can individually be mutatedto cysteine without interfering with expression of the protein orbinding to IL-2Rβγc. Positions were chosen to avoid interference withreceptor binding. FIG. 19B) Association kinetics of Neolukin-2/15cysteine mutants with IL-2βRγ_(c) measured using biolayerinterferometry. All of the variants associate with receptorapproximately similarly to Neo-2/15.

FIG. 20A-20C. Expression, purification, and thermal denaturationcharacterization of neoleukin-4. FIG. 20A) SDS Tris-Tricine gelelectrophoresis showing expression and purification over affinitycolumn. FIG. 20B) Circular dichroism at 222 nm during thermal meltingfrom 25° C. to 95° C., showing robust temperature stability. FIG. 20C)Circular dichroism wavelength scans at 25° C., 95° C. and then again 25°C., showing that neoleukin-4 does not fully melt at 95° C. and refoldsfully after cooling back to 25° C.

FIG. 21A-21D. Cytokine levels in non-human primates response to Neo-2/15or Neo-2/15-PEG. Two non-human primates (NHP) per group, one male andone female per group, were assigned to treatment with either vehicle,Neo-2/15 or Neo-2/15-PEG (comprising Neo-2/15 with a single cysteinemutation of E62C conjugated to PEG40K). Animals were administered either0 (vehicle), 0.1, 0.2 or 0.3 mg/kg of Neo-2/15, or 0.05, 0.10 or 0.15mg/kg of Neo-2/15-PEG, by intravenous bolus. Animals treated withNeo-2/15 PEG were administered by intravenous bolus. Cytokine sampleswere taken 0, 4, 8 and 24 hours post dose. Cytokine serum samples wereprepared and frozen at <−70° C. and shipped for analysis where sampleswere analyzed through a Luminex multiplex immunoassays system. Severalcytokines, including IL-10 (FIG. 21A-21B) and IL-15 (FIG. 21C-21D)demonstrated marked differences in the time course of cytokineproduction, consistent with a more sustained pharmacodynamic effect forthe PEGylated molecule.

FIG. 22: A block diagram of an example computing network.

FIG. 23A: A block diagram of an example computing device.

FIG. 23B: A block diagram of an example network of computing devicesarranged as a cloud-based server system.

FIG. 24: A flowchart of a method.

DETAILED DESCRIPTION

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”. Unlessotherwise required by context, singular terms used herein shall includepluralities and plural terms shall include the singular.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural or singular number, respectively.Additionally, the words “herein,” “above” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of thisapplication.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine(Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q),glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In one aspect, the invention provides non-naturally occurringpolypeptides comprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence at least 25%identical to EHALYDAL (SEQ ID NO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence at least 25%identical to YAFNFELI (SEQ ID NO:2);

(d) X4 is a peptide comprising the amino acid sequence at least 25%identical to ITILQSWIF (SEQ ID NO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and

wherein the polypeptide binds to IL-2 receptor βν_(c) heterodimer(IL-2Rβν_(c)) IL-4 receptor αν_(c) heterodimer (IL-4Rαν_(c)), or IL-13receptor α subunit (IL-13Rα). In various embodiments, the polypeptidesbind IL-2Rβν_(c) or IL-4Rαν_(c) with a binding affinity of 200 nM orless, 100 nM or less, 50 nM or less or 25 nM or less.

In one aspect, the invention provides non-naturally occurringpolypeptides comprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence at least 85%identical to EHALYDAL (SEQ ID NO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence at least 85%identical to YAFNFELI (SEQ ID NO:2);

(d) X4 is a peptide comprising the amino acid sequence at least 85%identical to ITILQSWIF (SEQ ID NO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and

wherein the polypeptide binds to IL-2 receptor βν_(c) heterodimer(IL-2Rβν_(c)). In various embodiments, the polypeptides bind IL-2Rβν_(c)with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or lessor 25 nM or less.

In one aspect, the invention provides non-naturally occurringpolypeptides comprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence EHALYDAL (SEQ IDNO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ IDNO:2);

(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ IDNO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains;and

wherein the polypeptide binds to IL-2 receptor βν_(c) heterodimer(IL-2Rβν_(c)). In various embodiments, the polypeptides bind IL-2Rβν_(c)with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or lessor 25 nM or less.

As shown in the examples that follow, the polypeptides of the disclosureare (a) mimetics of IL-2 and interleukin-15 (IL-15) that bind to theIL-2 receptor βν_(c) heterodimer (IL-2Rβν_(c)), but have no binding sitefor IL-2Rα or IL-15Rα, or (b) mimetics of IL-4 that bind to the IL-4receptor αν_(c) heterodimer (IL-4Rαν_(c)) or IL-13 receptor α subunit(IL-13Rα) (natural IL-4 and the IL-4 mimetics described hereincross-react with IL-13 receptor, forming an IL-4Rα/IL13Rα heterodimer).The designs are hyper-stable, bind to human and mouse IL-2Rβν_(c) orIL-4Rαν_(c) with higher affinity than the natural cytokines, and elicitdownstream cell signaling independent of IL-2Rα and IL-15Rα, orindependent of IL-13Rα. The polypeptides can be used, for example, totreat cancer.

The term protein mimetic as used herein refers to a protein thatimitates certain aspects of the function of another protein. The twoproteins typically have different amino acid sequence and/or differentstructures. Provided herein, among other things, are de novo mimetics ofIL-2 and IL-15. The aspects of the function of IL-2 and IL-15 that thesemimetics imitate is the induction of heterodimerization of IL-2Rβν_(c),leading to phosphorylation of STAT5. Because IL-2 and IL-15 both signalthrough heterodimerization of IL-2Rβν_(c), these mimetics imitate thisbiological function of both IL-2 and IL-15. These mimetics may bereferred to herein as mimetics of IL-2, of IL-15, or of both IL-2 andIL-15.

Also provided are de novo mimetics of IL-4. These mimetics are capableof imitating certain functions of IL-4. The function of IL-4 that thesemimetics imitate is the induction of heterodimerization of IL-4Rαν_(c)(and/or heterodimerization of IL-4Rα/IL-13Rα).

Native hIL-2 comprises four helices connected by long irregular loops.The N-terminal helix (H1) interacts with both the beta and gammasubunits, the third helix (H3) interacts with the beta subunit, and theC-terminal helix (H4) with the gamma subunit; the alpha subunitinteracting surface is formed by the irregular second helix (H2) and twolong loops, one connecting H1 to H2 and the other connecting H3 and H4.Idealized proteins were designed and produced in which H1, H3 and H4 arereplaced by idealized structural domains, including but not limited tohelices and beta strands (referred to as domains X1, X3 and X4,respectively) displaying an IL-2Rβν_(c) or IL-4Rαν_(c) interfaceinspired by H1, H3 and H4, and in which H2 is replaced with an idealizedhelix (referred to as domain X2) that offers better packing. As shown inthe examples, extensive mutational studies have been carried out,demonstrating that the amino acid sequence of each peptide domain eachcan be extensively modified without loss of binding to the IL-2 or IL-4receptor, and that the domains can be placed in any order whileretaining binding to the IL-2 or IL-4 receptor. The polypeptides maycomprise L amino acids and glycine, D-amino acids and glycine, orcombinations thereof.

Thus, X1, X2, X3, and X4 may be in any order in the polypeptide; innon-limiting embodiments, the ordering may be X1-X2-X3-X4; X1-X3-X2-X4;X1-X4-X2-X3; X3-X2-X1-X4; X4-X3-X2-X1; X2-X3-X4-X1; X2-X1-X4-X3; etc.

The domains may be separated by amino acid linkers of any length ofamino acid composition. There is no requirement for linkers; in oneembodiment there are no linkers present between any of the domains. Inother embodiments, an amino acid linker may be present between 1, 2, orall 3 junctions between domains X1, X2, X3, and X4. The linker may be ofany length as deemed appropriate for an intended use.

In various embodiments, X1 is a peptide comprising the amino acidsequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO:1. In other embodiments, X3 is a peptidecomprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95% m 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2. In furtherembodiments, X4 is a peptide comprising the amino acid sequence at least25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO:3.

In one embodiment, the polypeptides are IL-2/15 mimetics and (i) X1includes one or both of the following: H at residue 2 and Y at residue5; and/or (ii) X3 includes 1, 2, 3, 4, or all 5 of the following: Y atresidue 1, F at residue 3, N at residue 4, L at residue 7, and I atresidue 8. In a further embodiment, (iii) X4 includes I at residue 8.

In another embodiment, the polypeptides are IL-4 mimetics, and (i) X1includes E at residue 2 and K at residue 5; and (ii) X3 includes F atresidue 1, K at residue 3, R at residue 4, R at residue 7, and N atresidue 8. In a further embodiment, (iii) X4 includes F at residue 8.

In all of these embodiments, X1, X3, and X4 may be any suitable length,meaning each domain may contain any suitable number of additional aminoacids other than the peptides of SEQ ID NOS:1, 2, and 3, respectively.In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along itslength the peptide LEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is apeptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%identical along its length to the peptide EDEQEEMANAIITILQSWIFS (SEQ IDNO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 80% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 80% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 80% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS(SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 85% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 85% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 85% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS(SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 90% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 90% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 90% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS(SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence atleast 95% identical along its length to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 95% identical along its length the peptideLEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptide comprising theamino acid sequence at least 95% identical along its length to thepeptide EDEQEEMANAIITILQSWIFS(SEQ ID NO:6).

In one embodiment, X1 is a peptide comprising the amino acid sequence100% identical along its length to the peptide PKKKIQLHAEHALYDALMILNI(SEQ ID NO: 4); X3 is a peptide comprising the amino acid sequence 100%identical along its length to the peptide LEDYAFNFELILEEIARLFESG (SEQ IDNO:5); and X4 is a peptide comprising the amino acid sequence 100%identical along its length to the peptide EDEQEEMANAIITILQSWIFS(SEQ IDNO:6).

In one embodiment, the polypeptides are IL-2/15 mimetics and (i) X1includes 1, 2, 3, 4, or all 5 of the following: L at residue 7, H atresidue 8, H at residue 11, Y at residue 14; M at residue 18; and/or(ii) X3 includes 1, 2, 3, 4, 5, 6, 7, or all 8 of the following: D atresidue 3, Y at residue 4, F at residue 6, N at residue 7, L at residue10, I at residue 11, Eat residue 13, or E at residue 14. In a furtherembodiment, (iii) X4 includes I at residue 19.

In one embodiment of IL-2 mimetics, amino acid substitutions relative tothe reference peptide domains (i.e.: SEQ ID NOS: 1, 2, 3, 4, 5, or 6) donot occur at AA residues marked in bold font.

In another embodiment, the polypeptides are IL-4/IL-13 mimetics, and X1is a peptide comprising the amino acid sequence at least 25%, 27%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or100% identical along its length to the peptide

(SEQ ID NO: 8) PKKKIQIMA EEALKDAL SILNI;

X3 is a peptide comprising the amino acid sequence at least 37% 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%identical along its length the peptide

(SEQ ID NO: 9) LER FAKRFERN LWGIARLFESG;and

X4 is a peptide comprising the amino acid sequence at least 25%, 27%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 100% identical along its length to the peptide

(SEQ ID NO: 10) EDEQEEMANA IITILQSWFF S.

wherein

(i) X1 includes I at residue 7, T or M at residue 8, E at residue 11, Kat residue 14 and S at residue 18; and

(ii) X3 includes R at residue 3, F at residue 4, K at residue 6, R atresidue 7, R at residue 10, N at residue 11, W at residue 13, and G atresidue 14.

In a further embodiment, (iii) X4 includes F at residue 19.

In one embodiment, amino acid substitutions relative to the referencepeptide domains are conservative amino acid substitutions. As usedherein, “conservative amino acid substitution” means a given amino acidcan be replaced by a residue having similar physiochemicalcharacteristics, e.g., substituting one aliphatic residue for another(such as Ile, Val, Leu, or Ala for one another), or substitution of onepolar residue for another (such as between Lys and Arg; Glu and Asp; orGln and Asn). Other such conservative substitutions, e.g., substitutionsof entire regions having similar hydrophobicity characteristics, areknown. Polypeptides comprising conservative amino acid substitutions canbe tested in any one of the assays described herein to confirm that adesired activity, e.g. antigen-binding activity and specificity of anative or reference polypeptide is retained. Amino acids can be groupedaccording to similarities in the properties of their side chains (in A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers,New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro(P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S),Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu(E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturallyoccurring residues can be divided into groups based on common side-chainproperties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2)neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4)basic: His, Lys, Arg; (5) residues that influence chain orientation:Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutionswill entail exchanging a member of one of these classes for anotherclass. Particular conservative substitutions include, for example; Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into H is; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

In one embodiment, amino acid residues in X1 relative to SEQ ID NO:4 areselected from the group consisting of:

Position 01: A F I L M P Q R S W Position 02: A D E G V K Position 03: DE F W K Position 04: D E K N P R W Position 05: D E H I K L M S Position06: A D E G L P S W Q Position 07: D E L Q Y I Position 08: A F H W Y MT Position 09: C F P A Position 10: C D E F K P Position 11: D F H EPosition 12: A D E P S T V Position 13: H I L M P R V W Position 14: F RW Y K Position 15: D E N Y Position 16: A C L M S Position 17: F I L M PR Position 18: G M Q Y S Position 19: I L M P Q V Position 20: A K L M QR S Position 21: G K N P R S W Position 22: D E I K M N W Y

In one embodiment the polypeptides are IL-4 mimetics, and position 7 isI, position 8 is M or T, position 11 is E, position 14 is K, andposition 18 is S.

In another embodiment the polypeptides are IL-2 mimetics, and 1, 2, 3,4, or 5 of the following are not true: position 7 is I, position 8 is Mor T, position 11 is E, position 14 is K, and position 18 is S.

In another embodiment, amino acid residues in X3 relative to SEQ ID NO:5are selected from the group consisting of:

Position 01: A L Position 02: D E G K M T Position 03: D E N Y RPosition 04: C D G T Y F Position 05: A F H S V W Y Position 06: A F I MT V Y K Position 07: D K N S T R Position 08: A C G L M S V F Position09: C H K L R S T V E Position 10: F I L M Y R Position 11: I L N T YPosition 12: F K L M S V Position 13: A D F G I N P Q S T E W Position14: A E F G H S V Position 15: C I L M V W Position 16: A D G S T VPosition 17: H K L N R Position 18: C D G I L Q R T W Position 19: D F MN W Position 20: A C E F G M S Y Position 21: D E G H L M R S T V WPosition 22: A D G K N S Y

In another embodiment, the polypeptides are IL-4/IL-13 mimetics andposition 3 is R, position 4 is F, position 6 is K, position 7 is R,position 10 is R, position 11 is N, position 13 is W, and position 14 isG.

In another embodiment, the polypeptides are IL-2 mimetics and 1, 2, 3,4, 5, 6, 7, or all 8 of the following are not true: position 3 is R,position 4 is F, position 6 is K, position 7 is R, position 10 is R,position 11 is N, position 13 is W, and position 14 is G.

In any of such embodiments, the polypeptide further allows for acysteine at position 17 relative to SEQ ID NO:5 in addition to the aminoacid residues of H, K, L, N and R. Accordingly, amino acid residues inX3 relative to SEQ ID NO:5 can be selected from the group consisting of:

Position 01: A L Position 02: D E G K M T Position 03: D E N Y RPosition 04: C D G T Y F Position 05: A F H S V W Y Position 06: A F I MT V Y K Position 07: D K N S T R Position 08: A C G L M S V F Position09: C H K L R S T V E Position 10: F I L M Y R Position 11: I L N T YPosition 12: F K L M S V Position 13: A D F G I N P Q S T E W Position14: A E F G H S V Position 15: C I L M V W Position 16: A D G S T VPosition 17: H K L N R C Position 18: C D G I L Q R T W Position 19: D FM N W Position 20: A C E F G M S Y Position 21: D E G H L M R S T V WPosition 22: A D G K N S Y

In another embodiment, amino acid residues in X4 relative to SEQ ID NO:6are selected from the group consisting of:

Position 01: D E G K V Position 02: D I M S Position 03: E G H KPosition 04: E G I K Q R S Position 05: A D E G H S V Position 06: C D EG I M Q R T V Position 07: C E L M P R T Position 08: A F L M W Position09: A G L N Q R T Position 10: A C D E F H I W Position 11: I M N S V WPosition 12: I K L S V Position 13: C L M R S T Position 14: I L P T YPosition 15: F G I L M N V Position 16: H K Q R Position 17: C F K S W YPosition 18: K Q T W Position 19: C G N I Position 20: C F G L YPosition 21: A F G H S Y

In another embodiment, the polypeptides are IL-4/IL-13 mimetics andposition 19 is I. In another embodiment, the polypeptides are IL-2mimetics and position 19 is not I.

In any of such embodiments, the polypeptide further allows for acysteine at position 3 relative to SEQ ID NO:6 in addition to the aminoacid residues of E, G, H and K.

Accordingly, amino acid residues in X4 relative to SEQ ID NO:6 can beselected from the group consisting of:

Position 01: D E G K V Position 02: D I M S Position 03: E G H K CPosition 04: E G I K Q R S Position 05: A D E G H S V Position 06: C D EG I M Q R T V Position 07: C E L M P R T Position 08: A F L M W Position09: A G L N Q R T Position 10: A C D E F H I W Position 11: I M N S V WPosition 12: I K L S V Position 13: C L M R S T Position 14: I L P T YPosition 15: F G I L M N V Position 16: H K Q R Position 17: C F K S W YPosition 18: K Q T W Position 19: C G N I Position 20: C F G L YPosition 21: A F G H S Y

As noted herein, domain X2 is a structural domain, and thus any aminoacid sequence that connects the relevant other domains (depending ondomain order) and allows them to fold can be used. The length requiredwill depend on the structure of the protein being made and can be 8amino acids or longer. In one exemplary and non-limiting embodiment, X2is a peptide comprising the amino acid sequence at least 20%, 27%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical along itslength to KDEAEKAKRMKEWMKRIKT (SEQ ID NO:7). In a further embodiment,amino acid residues in X2 relative to SEQ ID NO:7 are selected from thegroup consisting of:

Position 01: A H L M R S V K Position 02: A D E Q R S T V W Y Position03: C E G K L N Q R W Position 04: A F G N S T V Y Position 05: A E G IM R V Position 06: C E K L N R V Position 07: A C E I L S T V W Position08: H K L M S T W Y Position 09: A I L M Q S R Position 10: A I M S W YPosition 11: C I K L S V Position 12: C E K L P Q R T Position 13: A D HN W Position 14: A C G I L S T V M Position 15: A E G I K L M R VPosition 16: G H L R S T V Position 17: A I L V Position 18: A C D E G HI K M S Position 19: D E G L N V T

In another embodiment, the polypeptides are IL-4/IL-13 mimetics andposition 11 is I. In another embodiment, the polypeptides are IL-2mimetics and position 11 is not I.

In any of such embodiments, the polypeptide further allows for acysteine at positions 5 or 16 relative to SEQ ID NO:7.

Alternatively, in any of such embodiments, the polypeptide furtherallows for a cysteine at positions 1, 2, 5, 9 or 16 relative to SEQ IDNO:7

Accordingly, amino acid residues in X2 relative to SEQ ID NO:7 can beselected from the group consisting of:

Position 01: A H L M R S V K C Position 02: A D E Q R S T V W Y CPosition 03: C E G K L N Q R W Position 04: A F G N S T V Y Position 05:A E G I M R V C Position 06: C E K L N R V Position 07: A C E I L S T VW Position 08: H K L M S T W Y Position 09: A I L M Q S R C Position 10:A I M S W Y Position 11: C I K L S V Position 12: C E K L P Q R TPosition 13: A D H N W Position 14: A C G I L S T V M Position 15: A E GI K L M R V Position 16: G H L R S T V C Position 17: A I L V Position18: A C D E G H I K M S Position 19: D E G L N V T

In another embodiment, the polypeptide comprises a polypeptide at leastat least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical along its length to the amino acid sequence selected from thegroup consisting of the following polypeptides (i.e.: SEQ ID NOS:11-94,103-184, 190-243, and 245-247). Underlined residues are linkers and areoptional and each residue of the linker, when present, may comprise anyamino acid. For each variant below, two SEQ ID NOS are provided: a firstSEQ ID NO: that includes the linker positions as optional and variable,and a second SEQ ID NO: that lists the sequence as shown below.

G1_neo2_33 H1->H4-STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDLDKAEDIRRNSDQARR >H2′-EAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 11) >H3STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDLDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 103) G1_neo2_34 H1->H4-STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSCISTGKCDLDKAEDIRRNSDQARR >H2′-EAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 12) >H3STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSCISTGKCDLDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAR (SEQ ID NO: 104) G1_neo2_35 H1->H4-STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDCDKAEDIRRNSDQARR >H2′-EAEKRGIDVRDLISNAQVILLEAC (SEQ ID NO: 13) >H3STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDCDKAEDIRRNSDQARREAEKRGIDVRDLISNAQVILLEAC (SEQ ID NO: 105) G1_neo2_36 H1->H4-STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELARNLEKVRD >H2′-EALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 14) >H3STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 106) G1_neo2_37 H1->H4-STKKLQLQAEHFLLDVQMILNESPEPNEELNRCITDAQSWISTGKIDLDRAEECARNLEKVRD >H2′-EALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 15) >H3STKKLQLQAEHFLLDVQMILNESPEPNEELNRCITDAQSWISTGKIDLDRAEECARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 107) G1_neo2_38 H1->H4-STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSCISTGKCDLDRAEELARNLEKVRD >H2′-EALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 16) >H3STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSCISTGKCDLDRAEELARNLEKVRDEALKRGIDVRDLVSNAKVIALELK (SEQ ID NO: 108) G1_neo2_39 H1->H4-STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELCRNLEKVRD >H2′-EALKRGIDVRDLVSNACVIALELK (SEQ ID NO: 17) >H3STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELCRNLEKVRDEALKRGIDVRDLVSNACVIALELK (SEQ ID NO: 109) G1_neo2_40 H1->H4-STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSWISTGKIDLDGAKELAKEVEELRQ >H2′-EAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 18) >H3STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSWISTGKIDLDGAKELAKEVEELRQEAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 110) G1_neo2_41 H1->H4-STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSCISTGKCDLDGAKELAKEVEELRQ >H2′-EAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 19) >H3STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSCISTGKCDLDGAKELAKEVEELRQEAEKRGIDVRDLASNLKVILLELA (SEQ ID NO: 111) G1_neo2_42 H1->H4-STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMAKEAEKIRK >H2′-EMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 20) >H3STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMAKEAEKIRKEMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 112) G1_neo2_43 H1->H4-STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSCISTGKCDLDNAQEMAKEAEKIRK >H2′-EMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 21) >H3STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSCISTGKCDLDNAQEMAKEAEKIRKEMEKRGIDVRDLISNIIVILLELS (SEQ ID NO: 113) G1_neo2_44 H1->H4-STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMCKEAEKIRK >H2′-EMEKRGIDVRDLISNICVILLELS (SEQ ID NO: 22) >H3STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMCKEAEKIRKEMEKRGIDVRDLISNICVILLELS (SEQ ID NO: 114) G1_neo2_40_1A H1->H4-STKKTQLLAEHALLDAFMMLNVVPEPNEKLNRIITTMQSWIYTGKIDADGAKELAKEVEELEQE >H2′-YEKRGIDVEDDASNLKVILLELA (SEQ ID NO: 23) >H3STKKTQLLAEHALLDAFMMLNVVPEPNEKLNRIITTMQSWIYTGKIDADGAKELAKEVEELEQEYEKRGIDVEDDASNLKVILLELA (SEQ ID NO: 115) G1_neo2_40_1B H1->H4-STKKTQLLAEHALLDAHMMLNMLPEPNEKLNRIITTMQSWIHTGKIDGDGAQELAKEVEELEQE >H2′-YEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 24) >H3STKKTQLLAEHALLDAHMMLNMLPEPNEKLNRIITTMQSWIHTGKIDGDGAQELAKEVEELEQEYEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 116) G1_neo2_40_1C H1->H4-STKKTQLLAEHALLDAFMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQE >H2′-FEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 25) >H3STKKTQLLAEHALLDAFMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQEFEKRGIDVEDEASNLKVILLELA (SEQ ID NO: 117) G1_neo2_40_1D H1->H4-STKKTQLLAEHALLDALMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQE >H2′-LEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 26) >H3STKKTQLLAEHALLDALMMLNMVPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQELEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 118) G1_neo2_40_1E H1->H4-STKKTQLLAEHALLDAHMMLNVVPEPNEKLNRIITTMQSIYTGKIDRDGAQELAKEVEELEQE >H2′-LEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 27) >H3STKKTQLLAEHALLDAHMMLNVVPEPNEKLNRIITTMQSWIYTGKIDRDGAQELAKEVEELEQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 119) G1_neo2_40_1F H1->H4-STKKTQLLAEHALLDALMMLNLLPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQE >H2′-HEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 28) >H3STKKTQLLAEHALLDALMMLNLLPEPNEKLNRIITTMQSWIFTGKIDGDGAQELAKEVEELEQEHEKRGIDVEDYASNLKVILLELA (SEQ ID NO: 120) G1_neo2_40_1G H1->H4-STKKTQLLAEHALLDAYMMLNMVPEPNEKLNRIITTMQSWILTGKIDSDGAQELAKEVEELEQE >H2′-LEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 29) >H3STKKTQLLAEHALLDAYMMLNMVPEPNEKLNRIITTMQSWILTGKIDSDGAQELAKEVEELEQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 121) G1_neo2_40_1H H1->H4-STKKTHLLAEHALLDAYMMLNVMPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQE >H2′-FEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 30) >H3STKKTHLLAEHALLDAYMMLNVMPEPNEKLNRIITTMQSWIFTGKIDGDGAKELAKEVEELEQEFEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 122) G1_neo2_40_1I H1->H4-STKKTQLLAEHALLDAYMMLNLVPEPNEKLNRIITTMQSWIFTGKIDADGAQELAIEVEELEQE >H2′-YEKRGIDVDDYASNLKVILLELA (SEQ ID NO: 31) >H3STKKTQLLAEHALLDAYMMLNLVPEPNEKLNRIITTMQSWIFTGKIDADGAQELAIEVEELEQEYEKRGIDVDDYASNLKVILLELA (SEQ ID NO: 123) G1_neo2_40_1J H1->H4-STKKTQLMAEHALLDAFMMLNVLPEPNEKLNRIITTMQSWIFTGKIDGDDAQELAKEVEELEQE >H2′-LEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 32) >H3STKKTQLMAEHALLDAFMMLNVLPEPNEKLNRIITTMQSWIFTGKIDGDDAQELAKEVEELEQELEKRGIDVDDDASNLKVILLELA (SEQ ID NO: 124) G1_neo2_40_1F_H1 H1->H4-STKKTQLLIEHALLDALDMSRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQQLAKEVEELEQE >H2′-HEKRGEDVEDEASNLKVILLELA (SEQ ID NO: 33) >H3STKKTQLLIEHALLDALDMSRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQQLAKEVEELEQEHEKRGEDVEDEASNLKVILLELA (SEQ ID NO: 125) G1_neo2_40_1F_H2 H1->H4-STKKTQLLLEHALLDALHMRRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQELAKEVEELEQE >H2′-HEKRGRDVEDDASNLKVILLELA (SEQ ID NO: 34) >H3STKKTQLLLEHALLDALHMRRNLPEPNEKLSRIITTMQSWIFTGKIDGDGAQELAKEVEELEQEHEKRGRDVEDDASNLKVILLELA (SEQ ID NO: 126) G1_neo2_40_1F_H3 H1->H4-STKKTQLLIEHALLDALNMRKKLPEPNEKLSRIITDMQSWIFTGKIDGDGAQQLAKEVEELEQE >H2′-HEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 35) >H3STKKTQLLIEHALLDALNMRKKLPEPNEKLSRIITDMQSWIFTGKIDGDGAQQLAKEVEELEQEHEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 127) G1_neo2_40_1F_H4 H1->H4-STKKTQLLLEHALLDALHMSRELPEPNEKLNRIITDMQSWIFTGKIDGDGAQDLAKEVEELEQE >H2′-HEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 36) >H3STKKTQLLLEHALLDALHMSRELPEPNEKLNRIITDMQSWIFTGKIDGDGAQDLAKEVEELEQEHEKRGGDVEDYASNLKVILLELA (SEQ ID NO: 128) G1_neo2_40_1F_H5 H1->H4-STKKTQLLIEHALLDALHMSRKLPEPNEKLSRIITTMQSWIFTGKIDGDGAQHLAKEVEELEQE >H2′-HEKRGGEVEDEASNLKVILLELA (SEQ ID NO: 37) >H3STKKTQLLIEHALLDALHMSRKLPEPNEKLSRIITTMQSWIFTGKIDGDGAQHLAKEVEELEQEHEKRGGEVEDEASNLKVILLELA (SEQ ID NO: 129) G1_neo2_40_1F_H6 H1->H4-STKKTQLLIEHALLDALHMKRKLPEPNEKLNRIITNMQSWIFTEKIDGDGAQDLAKEVEELEQE >H2′-HEKRGQDVEDYASNLKVILLELA (SEQ ID NO: 38) >H3STKKTQLLIEHALLDALHMKRKLPEPNEKLNRIITNMQSWIFTEKIDGDGAQDLAKEVEELEQEHEKRGQDVEDYASNLKVILLELA (SEQ ID NO: 130) G1_neo2_40_1F_M1 H1->H4-STEKTQLAAEHALRDALMLKHLLNEPNEKLARIITTMQSWQFTGKIDGDGAQELAKEVEELQQE >H2′-HEVRGIDVEDYASNLKVILLHLA (SEQ ID NO: 39) >H3STEKTQLAAEHALRDALMLKHLLNEPNEKLARIITTMQSWQFTGKIDGDGAQELAKEVEELQQEHEVRGIDVEDYASNLKVILLHLA (SEQ ID NO: 131) G1_neo2_40_1F_M2 H1->H4-STKNTQLAAEDALLDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQE >H2′-HEERGIDVEDYASNLKVILLQLA (SEQ ID NO: 40) >H3STKNTQLAAEDALLDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQEHEERGIDVEDYASNLKVILLQLA (SEQ ID NO: 132) G1_neo2_40_1F_M3 H1->H4-STEKTQHAAEDALRDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQE >H2′-HEVRGIDVEDYASNLKVILLQLA (SEQ ID NO: 41) >H3STEKTQHAAEDALRDALMLRNLLNEPNEKLARIITTMQSWQFTEKIDGDGAQELAKEVEELQQEHEVRGIDVEDYASNLKVILLQLA (SEQ ID NO: 133) G2_neo2_40_ H1->H4-TQKKQQLLAEHALLDALMILNMLKTSSEAVNRMITIAQSWIFTGTSNPEEAKEMIKMAEQAEEE1F_seq02 >H2′- ARREGVDTEDYVSNLKVILKEIA (SEQ ID NO: 42) >H3TQKKQQLLAEHALLDALMILNLDKTSSEAVNRMITIAQSWIFTGTSNPEEAKEMIKMAEQAEEEARREGVDTEDYVSNLKVILKEIA (SEQ ID NO: 134) G2_neo2_40_ H1->H4-TTKKYQLLVEHALLDALMMLNLSSESNEKMNRIITTMQSWIFTGTFDPDQAEELAKLVEELPEE1F_seq03 >H2′- FRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 43) >H3TTKKYQLLVEHALLDALMMLNLSSESNEKMNRIITTMQSWIFTGTFDPDQAEELAKLVEELREEFRKRGIDTEDYASNLKVILKELS (SEG ID NO: 135) G2_neo2_40_ H1->H4-TTKKIQLLVEHALLDALMILNLSSESNEKLNRIITTLQSWIFRGEIDPDRARELAKLLEEIREE1F_seq04 >H2′- MRKRGIDTEDYVSNMIVIIRELA (SEQ ID NO: 44) >H3TTKKIQLLVEHALLDALMILNLSSESNEKLNRIITTLQSWIFRGEIDPDRARELAKLLEEIREEMRKRGIDTEDYVSNMIVIIRELA (SEQ ID NO: 136) G2_neo2_40_ H1->H4-TKKKIQLLAEHVLLDLLMMLNLSSESNEKMNRLITIVQSWIFTGTIDPDQAEEMAKWVEELREE1F_seq05 >H2′- FRKRGIDTEDYASNVKVILKELS (SEQ ID NO: 45) >H3TKKKIQLLAEHVLLDLLMMLNLSSESNEKMNPLITIVQSWIFTGTIDPDQAEEMAKWVEELREEFRKRGIDTEDYIASNVKVIKELS SEQ ID NO: 137) G2_neo2_40_ H1->H4-TKKKYQLLIEHLLLDALMVLNMSSESNEKLNRIITILQSWIFTGTWDPDLAEEMEKLMQEIEEE1F_seq06 >H2′- LRRRGIDTEDYMSNMRVIIKELS (SEQ ID NO: 46) >H3TKKKYQLLIEHLLLDALMVLNMSSESNEKLNRIITILQSWIFTGTWDPDLAEEMEKLMQEIEEELRRRGIDTEDYMSNMRVIIKELS (SEQ ID NO: 138) G2_neo2_40_ H1->H4-TKKKLQLLVEHLLLDMLMILNMSSESNEKLNRLITELQSWIFRGEIDPDKAEEMWKIMEEIEKE1F_seq07 >H2′- LRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 47) >H3TKKKLQLLVEHLLLDMLMILNMSSESNEKLNRLITELQSWIFRGEIDPDKAEEMWKIMEEIEKELRERGIDTEDYMSNAKVIIKELS SEQ ID NO: 139) G2_neo2_40_ H1->H4-TSKKQQLLAEHALLDALMILNISSESSEAVNRAITWLQSWIFKGTVNPDQAEEMRKLAEQIREE1F_seq08 >H2′- MRKRGIDTEDYVSNLEVIAKELS (SEQ ID NO: 48) >H3TSKKQQLLAEHALLDALMILNISSESSEAVNRAITWLQSWIFKGTVNPDQAEEMRKLAEQIREEMRKRGIDTEDYVSNLEVIAKELS (SEQ ID NO: 140) G2_neo2_40_ H1->H4-TKKKYQLLIEHLLLDLLMVLNMSSESNEKINPLITWLQSWIFTGTYDPDLAEEMYKILEELREE1F_seq09 >H2′- MRERGIDTEDYMSNMRVIVKELS (SEQ ID NO: 49) >H3TKKKYQLLIEHLLLDLLMVLNMSSESNEKINPLITWLQSWIFTGTYDPDLAEEMYKILEELREEMRERGIDTEDYMSNMRVIVKE-LS (SEQ ID NO: 141) G2_neo2_40_ H1->H4-TKKKWQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFTGTYDPDLAEEMKKMMDEIEDE1F_seq10 >H2′- LRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 50) >H3TKKKWQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFTGTYDPDLAEEMKKMMDEIEDELRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 142) G2_neo2_40_ H1->H4-TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELSKLVEEIREE1F_seq11 >H2′- MRKRGIDTEDYVSNLKVILDELS (SEQ ID NO: 51) >H3TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELSKLVEEIREEMRKRGIDTEDYVSNLKVILDELS (SEQ ID NO: 113) G2_neo2_40_ H1->H4-TEKKLQLLVEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGRIDPDKAEELAKLVEELREE1F_seq12 >H2′- ARERGIDTEDYVSNLKVILKELS (SEQ ID NO: 52) >H3TEKKLQLLVEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGRIDPDKAEELAKLVEELREEARERGIDTEDYVSNLKVILKELS (SEQ ID NO: 144) G2_neo2_40_ H1->H4-TKKKYQLLMEHLLLDLLMVLNMSSESNEKLNRLITIIQSWIFTGTWDPDKAEEMAKMLKEIEDE1F_seq13 >H2′- LRERGIDTEDYMSNMIVIMKELS (SEQ ID NO: 53) >H3TKKKYQLLMEHLLLDLLMVLNMSSESNEKLNRLITIIQSWIFTGTWDPDKAEEMAKMLKEIEDELRERGIDTEDYMSNMIVIMKELS (SEQ ID NO: 115) G2_neo2_40_ H1->H4-TTKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFEGRIDPDQAQELAKLVEELREE1F_seq14 >H2′- FRKRGIDTEDYVSNLKVILEELS (SEQ ID NO: 54) >H3TTKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFEGRIDPDQAQELAKLVEELREEFRKRGIDTEDYVSNLKVILEELS (SEQ ID NO: 146) G2_neo2_40_ H1->H4-TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELAKLVRELREE1F_seq15 >H2′- FRKRGIDTEDYASNLEVILRELS (SEQ ID NO: 55) >H3TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDQAEELAKLVRELREEFRKRGIDTEDYASNLEVILRELS (SEQ ID NO: 147) G2_neo2_40_ H1->H4-TKKKIQLLVEHALLDALMILNLSSKSNEKLNRIITTMQSWIFNGTIDPDRARELAKLVEEIRDE1F_seq16 >H2′- MEKNGIDTEDYVSNLKVILEELA (SEQ ID NO: 56) >H3TKKKIQLLVEHALLDALMILNLSSKSNEKLNRIITTMQSWIFNGTIDPDRARELAKLVEEIRDEMEKNGIDTEDYVSNLKVILEELA (SEQ ID NO: 148) G2_neo2_40_ H1->H4-TKKKYQLLIEHVLLDLLMLLNLSSESNEKMNRLITILQSWIFTGTYDPDKAEEMAKLLKELREE1F_seq17 >H2′- FRERGIDTEDYISNAIVILKELS (SEQ ID NO: 57) >H3TKKKYQLLIEHVLLDLLMLLNLSSESNEKMNRLITILQSWIFTGTYDPDKAEEMAKLLKELREEFRERGIDTEDYISNAIVILKELS (SEQ ID NO: 149) G2_neo2_40_ H1->H4-TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDRAEELAKLVEELREE1F_seq18 >H2′- FRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 58) >H3TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFTGTIDPDRAEELAKLVEELREEFRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 150) G2_neo2_40_ H1->H4-TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFNGTIDPDQARELAKLVEELREE1F_seq19 >H2′- FRKRGIDTEDYASNLKVILEELA (SEQ ID NO: 59) >H3TKKKIQLLVEHALLDALMMLNLSSESNEKLNRIITTMQSWIFNGTIDPDQARELAKLVEELREE FRKRGIDTEDYASNLKVILEELA (SEQ ID NO: 151) G2_neo2_40_ H1->H4-TKKKLQLLVEHALLDALMLLNLSSESNEKLNRIITTMQSWIFTGTVDPDQAEELAKLVEEIREE1F_seq20 >H2′- LRKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 60) >H3TKKKLQLLVEHALLDALMLLNLSSESNEKLNRIITTMQSWIFTGTVDPDQAEELAKLVEEIREELRKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 152)  G2_neo2_40 H1->H4-TTKKYQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTFDPDQAEELAKLVREIREE1F_seq21 >H2′- MRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 61) >H3TTKKYQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTFDPDQAEELAKLVREIREEMRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 153)   G2_neo2_40_ H1->H4-TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDPDRAEELAKLVREIREE1F_seq22 >H2′- LRKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 62) >H3TKKKIQLLVEHALLDALMILNLSSESNEKLNRIITTMQSWIFTGTIDPDRAEELAKLVREIREELRKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 152)   G2_neo2_40_ H1->H4-TKKKYQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFRGEWDPDKAEEWAKILKEIREE1F_seq23 >H2′- LRERGIDTEDYMSNAIVIMKELS (SEQ ID NO: 63) >H3TKKKYQLLIEHLLLDLLMILNLSSESNEKLNRLITWLQSWIFRGEWDPDKAEEWAKILKEIREELRERGIDTEDYMSNAIVIMKELS (SEQ ID NO: 155) G2_neo2_40_ H1->H4-TDKKLQLLVEHLLLDLLMMLNLSSKSNEKMNRLITIAQSWIFTGKVDPDLAREMIKLLEETEDE1F_seq24 >H2′- NPKNGIDTEDYVSNARVIAKELE (SEQ ID NO: 64) >H3TDKKLQLLVEHLLLDLLMMLNLSSKSNEKMNRLITIAQSWIFTGKVDPDLAREMIKLLEETEDENPKNGIDTEDYVSNARVIAKELE (SEG ID NO: 156) G2_neo2_40_ H1->H4-TKKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFTGTIDPDQAEELAKLVEELKEE1F_seq25 >H2′- FKKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 65) >H3TKKKIQLLVEHALLDALMLLNLSSESNEKMNRIITTMQSWIFTGTIDPDQAEELAKLVEELKEEFKKRGIDTEDYVSNLKVILKELS (SEQ ID NO: 157) G2_neo2_40_ H1->H4-TKKKYQLLIEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGTYDPDKAEELEKLAKEIEDE1F_seq26 >H2′- ARERGIDTEDYMSNLRVILKELS (SEQ ID NO: 66) >H3TKKKYQLLIEHALLDALMILNLWSESNEKLNRIITTMQSWIFTGTYDPDKAEELEKLAKEIEDEARERGIDTEDYMSNLRVILKELS (SEG ID NO: 158) G2_neo2_40_ H1->H4-TKKKAQLLAEHALLDALMLLNLSSESNERLNRIITWLQSIIFTGTYDPDMVKEAVKLADEIEDE1F_seq27 >H2′- MRKRGIDTEDYVSNLRVILQELA (SEQ ID NO: 67) >H3TKKKAQLLAEHALLDALMLLNLSSESNERLNRIITWLQSIIFTGTYDPDMVKEAVKLADEIEDEMRKRGIDTEDYVSNLRVILQELA (SEQ ID NO: 159) G2_neo2_40_ H1->H4-TQKKNQLLAEHLLLDALMVLNQSSESSEVANRIITWAQSWIFEGRVDPNKAEEAKKLAKKLEEE1F_seq28 >H2′- MRKRGIDMEDYISNMKVIAEEMS (SEQ ID NO: 68) >H3TQKKNQLLAEHLLLDALMVLNQSSESSEVANRIITWAQSWIFEGRVDPNKAEEAKKLAKKLEEEMRKRGIDMEDYISNMKVIAEEMS SEQ ID NO: 160) G2_neo2_40_ H3-EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYI1F_seq29 >E2′-QSQIFEISERIRETDQEKKEESWKKWQLLLEHALLDVLMLLND (SEQ ID NO: 69) >H4->H1EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEISERIRETDQEKKEESWKKWQLLLEHALLDVLMLLND (SEQ ID NO: 161) G2_neo2_40_H1->H3- PEKKRQLLLEHILLDALMLLNLXXXXXXNTESKFEDYISNAEVIAEELAKLMESXXLSDEAEKF1F_seq30 >H2′-KKIKQWLREVWRIWXXXXWSTLEDKARELLNRIITTIQSQIFY (SEQ ID NO: 70) >H4PEKKRQLLLEHILLDALMLLNLLETNPQNTESKFEDYISNAEVIAEELAKLMESLGLSDEAEKFKKIKQWLREVWRIWSSTNWSTLEDKARELLNRIITTIQSQIFY (SEQ ID NO: 162) G2_neo2_40_H1->H3- PEKKRQLLLEHILLDLLMILNMXXXXXXNTESEMEDYWSNVRVILRELARLMEEXXXKELSELM1F_seq31 >H2′-ERMRKIVEKIRQIVTXXXXLDTAREWLNRLITWIQSLIFR (SEQ ID NO: 71) >H4PEKKRQLLLEHILLDLLMILNMIETNRENTESEMEDYWSNVRVILRELARLMEELNYKELSELMERMRKIVEKIRQIVTNNSSLDTAREWLNRLITWIQSLIFR (SEQ ID NO: 163) G2_neo2_40_H1->H3- PEKKRQLLAEHALLDALMLLNIIETNSKNTESKMEDYVSNLEVILTEFKKLAEKLNFSEEAERA1F_seq32 >E2′-ERMKRWARKAYQMMTLDLSLDKAKEMLNRIITILQSIIFN (SEQ ID NO: 72) >H4PEKKRQLLAEHALLDALMLLNIIETNSKNTESKMEDYVSNLEVILTEFKKLAEKLNFSEEAERAERMKRWARKAYQMMTLDLSLDKAKEMLNRIITILQSIIFN (SEQ ID NO: 164) G2_neo2_40_H1->H3- PEKKRQLLAEHLLLDVLMMLNGNASLKDYASNAQVIADEFRELARELGLTDEAKKAEKIIEALE1F_seq33 >H2′- PAREWLLNNKDKE32EALNRAITIAQSWIFN (SEQ ID NO: 73) >H4PEKKRQLLAEHLLLDVLMMLNGNASLKDYASNAQVIADEFRELARELGLTDEAKKAEKIIEALEPAPNNKDKEKAKFAINPAITIAQSWIEN (SEQ ID NO: 165) G2_neo2_40_ H1->H3-PEKKRQLLLEHLLLDLLMILNMLRTNPKNIESDWEDYMSNIEVIIEELRKIMESLGRSEKAKEW1F_seq34 >H2′-KRMKQWVRRILEIVKNNSDLEEAKEWLNRLITIVQSEIFE (SEQ ID NO: 74) >H4PEKKRQLLLEHLLLDLLMILNMLRTNPKNIESDWEDYMSNIEVIIEELRKIMESLGRSEKAKEWKRMKQWVRRILEIVKNNSDLEEAKEWLNRLITIVQSEIFE (SEQ ID NO: 166) G2_neo2_40_H1->H3- WEKKRQLLLEHLLLDLLMILNMWRTNPQNTESLMEDYMSNAKVIVEELARMMRSQGLEDKAREW1F_seq35 >H2′-EEMKKRIEEIRQIIQNNSSKERAKEELNRLITYVQSEIFR (SEQ ID NO: 75) >H4WEKKRQLLLEHLLLDLLMILNMWRTNPQNTESLMEDYMSNAKVIVEELARMMRSQGLEDKAREWEEMKKRIEEIRQIIQNNSSKERAKEELNRLITYVQSEIFR (SEQ ID NO: 167) G2_neo2_40_H1->H3- PKKKIQLLAEHALLDALMILNIVKTNSQNAEEKLEDYASNVEVILEEIARLMESGDQKDEAEKA1F_seq36 >H2′- KRMKEWMKRIKTTASEDEQEEMANPIITLLQSWIFS (SEQ ID NO: 76) >H4PKKKIQLLAEHALLDALMILNIVKTNSQNAEEKLEDYASNVEVILEEIARLMESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANPIITLLQSWIFS (SEQ ID NO: 166) G2_neo2_40_H1->H3- PEKKRQLLAEHALLDALMILNXXXXXXQNAEEKLEDYMSNVEVIMEEFARMMRXXXXSEEAENA1F_seq37 >H2′- ERIKKWVRKASSXXXSEEQREMMNRAITLMQSWIFE (SEQ ID NO: 77) >H4PEKKRQLLAEHALLDALMILNILQTNPQNAEEKLEDYMSNVEVIMEEFARMMRNGDRSEEAENAERIKKWVRKASSTASSEEQREMMNRAITLMQSWIFE (SEQ ID NO: 169) G2_neo2_40_H1->H3- PEKKRQLLAEHLLLDALMVLNMXXXXXXNTEEKLEDYISNMKVIIKEMIELMRSLXXXEEAEKW1F_seq38 >H2′- KEALKAVEKIXXXXDSETARELANRIITLAQSAIFY (SEQ ID NO: 78) >H4PEKKRQLLAEHLLLDALMVLNMLTTNSKNTEEKLEDYISNMKVIIKEMIELMRSLGRLEEAEKWKEALKAVEKIGSRMDSETARELANRIITLAQSAIFY (SEQ ID NO: 170) G2_neo2_40_H1->H3- PEKKRQLLAEHALLDALMFLNLXXXXXXQAEEKIEDYASNLRVIAEELARLFENLXXXDEAQKA1F_seq39 >H2′- KDIKELAERARSXXSSEKRKEAMNRAITILQSMIFR (SEQ ID NO: 79) >H4PEKKRQLLAEHALLDALMFLNLVETNPDQAEEKIEDYASNLRVIAEELARLFENLGRLDEAQKAKDIKELAERARSRVSSEKRKEAMNRAITILQSMIFR (SEQ ID NO: 171) G2_neo2_40_H1->H3- PEKKRQLLAEHALLDALMILNIIRTNSDNTESKLEDYISNLKVILEEIARLMESLGLSDEAEKA1F_seq40 >E2′- KEAMRLADKAGSTASEEEKKEAMNRVITWAQSWIFN (SEQ ID NO: 80) >H4PEKKRQLLAEHALLDALMILNIIRTNSDNTESKLEDYISNLKVILEEIARLMESLGLSDEAEKAKEAMRLADKAGSTASEEEKKEAMNRVITWAQSWIFN (SEQ ID NO 172) G2_neo2_40_ H1->H3-PEKKRQLLAEHALLDALMMLNILRTNPDNAEEKLEDYWSNLIVILREIAKLMESLGLTDEAEKA1F_seq41 >H2′- KEAARWAEEARTTASKDQRRELANRIITLLQSWIFS (SEQ ID NO: 81) >H4PEKKRQLLAEHALLDALMMLNILRTNPDNAEEKLEDYWSNLIVILREIAKLMESLGLTDEAEKAKEAARWAEEARTTASKDQRRELANRIITLLQSWIFS (SEQ ID NO: 173) G2_neo2_40_H1->H3- PEKKRQLLAEHLLLDALMILNIIETNEQNAESKLEDYISNAKVILDEFREMARDLGLLDEAKKA1F_seq42 >H2′- EKMKRWLEKMRSNASSDERREWANRMITTAQSWIFN (SEQ ID NO: 82) >H4PEKKRQLLAEHLLLDALMILNIIETNEQNAESKLEDYISNAKVILDEFREMARDLGLLDEAKKAEKMKRWLEKMRSNASSDERREWANRMITTAQSWIFN (SEQ ID NO: 174) G2_neo2_40_H1->H4- TNKKAQLHAEFALHDALMLLNLSSESNERLNRIITWLQSIIFYGTYDPDMVKEAVKDADEIEDE1F_seq27_S3 >H2′- MRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 83) >H3TNKKAQLHAEFALHDALMLLNLSSESNERLNRIITWLQSIIFYGTYDPDMVKEAVKDADEIEDEMRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 245) G2_neo2_40_ H1->H4-TNKEAQLHAEFALYDALMLLNLSSESNERLNRIITWLQSIIFYETYDPDMVKEAVKLADEIEDE1F_seq27_S18 >H2′- MRKRKIDTEDYVVNLRLILQELA (SEQ ID NO: 84) >H3TNKEAQLHAEFALYDALMLLNLSSESNERLNRIITWLQSIIFYETYDPDMVKEAVKLADEIEDEMRKRKIDTEDYVVNLRLILQELA (SEQ ID NO: 175) G2_neo2_40_ H1->H4-TKKDAELLAEFALYDALMLLNLSSESNERLNEIITWLQSIIFYGTYDPDMVKEAVKLADEIEDE1F_seq27_S22 >H2′- MRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 85) >H3TKKDAELLAEFALYDALMLLNLSSESNERLNEIITWLQSIIFYGTYDPDMVKEAVKLADEIEDEMRKRGIDTEDYVSNLRLILQELA (SEQ ID NO: 176) G2_neo2_40_ H1->H4-TNKKAQLHAEFALYDALMLLNLSSESNERLNDIITWLQSIIFTGTYDPDMVKEAVKLADEIEDE1F_seq27_S24 >H2′- MRKRKIDTEDYVVNLRYILQELA (SEQ ID NO: 86) >H3TNKKAQLHAEFALYDALMLLNLSSESNERLNDIITWLQSIIFTGTYDPDMVKEAVKLADEIEDEMRKRKIDTEDYVVNLRYILQELA (SEQ ID NO: 177) G2_neo2_40_ H3-EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYI1F_seq29_S6 >H2′-QSQIFEVLHGVGETDQEKKEESWKKWDLLLEHALLDVLMLLND (SEQ ID NO: 87) >H4->H1EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEVLHGVGETDQEKKEESWKKWDLLLEHALLDVLMLLND (SEQ ID NO: 178) G2_neo2_40_H3- EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNELITYI1F_seq29_S7 >H2′-QSQIFEVIEREGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 88) >H4->H1EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNELITYIQSQIFEVIEREGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 179) G2_neo2_40_H3- EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYI1F_seq29_S8 >H2′-QSQIFEVLEGVGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 89) >H4->H1EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEVLEGVGETDQEKKEESWKKWELHLEHALLDVLMLLND (SEQ ID NO: 180) Neoleukin-H1->H3- PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKA2/15 >H2′- KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 90)(i.e. >H4PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAG2_neo2_40_ KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 181)1F_seq36_S11) G2_neo2_40_ H1->H3-PKKKIQLLAEHALFDLLMILNIVKTNSQNAEEKLEDYAYNAGVILEEIARLFESGDQKDEAEKA1F_seq36_S12 >H2′-KRMKEWMKRIKDTASEDEQEEMANEIITILQSWNFS (SEQ ID NO: 91) >H4PKKKIQLLAEHALFDLLMILNIVKTNSQNAEEKLEDYAYNAGVILEEIARLFESGDQKDEAEKAKRMKEWMKRIKDTASEDEQEEMANEIITILQSWNFS (SEQ ID NO: 182) Neoleukin- H1->H3-PKKKIQLYAEHALYDALMILNIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQKDEAEKA2/15-H8Y- >H2′- KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 94)K33E >H4PKKKIQLYAEHALYDALMILNIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 246) Neoleukin- H1->H3-PKKKIQLHAEHALYDALMILNIVKTNSPPAEEK LEDYAFNFELILEEIARLFESGDQKDEAEK2/15 >H2′- AKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 247)(K32 is >H4 considered to be a residue of the optional linker in thisdepicted sequence) IL4_G2_neo2_40_PKKKIQITAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMKE1F_seq36_S11 WMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 92)PKKKIQITAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 183) Neoleukin-4PKKKIQIMAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMIE(i.e. WMKRIKTTASEDEQEEMANAIITILQSWFFS (SEQ ID NO: 93) IL4_G2_neo2_40_PKKKIQIMAEEALKDALSILNIVKTNSPPAEEQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMIE1F_seq36_S11_ WMKRIKTTASEDEQEEMANAIITILQSWFFS (SEQ ID NO: 184) MIF)

For each variant below, two SEQ ID NOs are provided: a first SEQ ID NO:that lists the sequence as shown below, and a second SEQ ID NO: thatincludes the linker positions as optional and variable.

>Neoleukin-2/15_R50C (SEQ ID NO: 190)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C (SEQ ID NO: 217)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIACLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C(SEQ ID NO: 191)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C (SEQ ID NO: 218)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFCSG XX KDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C(SEQ ID NO: 192)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C (SEQ ID NO: 219)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C(SEQ ID NO: 193)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C (SEQ ID NO: 220)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX CDEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C(SEQ ID NO: 194)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C (SEQ ID NO: 221)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KCEAEKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C(SEQ ID NO: 195)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C (SEQ ID NO: 222)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEACKAKRMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C(SEQ ID NO: 196)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C (SEQ ID NO: 223)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKCMKEWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C(SEQ ID NO: 197)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKCWMKRIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C (SEQ ID NO: 224)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKCWMKRIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R73C(SEQ ID NO: 198)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R73C (SEQ ID NO: 225)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKCIKT XXX EDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_T77C(SEQ ID NO: 199)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTCASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_T77C (SEQ ID NO: 226)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKTCASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E82C (SEQ ID NO: 200)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E82C (SEQ ID NO: 227)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E85C(SEQ ID NO: 201)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQECMANAIITILQSWIFS* >Neoleukin-2/15_E85C (SEQ ID NO: 228)PKKKIQLHAEHALYDALMILNI XXXXXXXXXXX LEDYAFNFELILEEIARLFESG XX KDEAEKAKRMKEWMKRIKT XXX EDEQECMANAIITILQSWIFS* >Neoleukin-2/15_R50C_R73C(SEQ ID NO: 202)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C_R73C(SEQ ID NO: 229) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIACLFESG XX KDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_R73C (SEQ ID NO: 203)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_R73C(SEQ ID NO: 230) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFCSG XX KDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_R73C (SEQ ID NO: 204)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_R73C(SEQ ID NO: 231) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_R73C (SEQ ID NO: 205)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_R73C(SEQ ID NO: 232) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XXCDEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_R73C (SEQ ID NO: 206)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_R73C(SEQ ID NO: 233) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KCEAEKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_R73C (SEQ ID NO: 207)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_R73C(SEQ ID NO: 234) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEACKAKRMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_R73C (SEQ ID NO: 208)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_R73C(SEQ ID NO: 235) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEAEKAKCMK EWMKCIKT XXXEDEQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C_E82C (SEQ ID NO: 209)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_R50C_E82C(SEQ ID NO: 236) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIACLFESG XX KDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_E82C (SEQ ID NO: 210)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E53C_E82C(SEQ ID NO: 237) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFCSG XX KDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_E82C (SEQ ID NO: 211)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D56C_E82C(SEQ ID NO: 238) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_E82C (SEQ ID NO: 212)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_K58C_E82C(SEQ ID NO: 239) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XXCDEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_E82C (SEQ ID NO: 213)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_D59C_E82C(SEQ ID NO: 240) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KCEAEKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_E82C (SEQ ID NO: 214)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E62C_E82C(SEQ ID NO: 241) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEACKAKRMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_E82C (SEQ ID NO: 215)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_R66C_E82C(SEQ ID NO: 242) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEAEKAKCMK EWMKRIKT XXXEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C_E82C (SEQ ID NO: 216)PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKCWMKRIKTTASEDCQEEMANAIITILQSWIFS* >Neoleukin-2/15_E69C_E82C(SEQ ID NO: 243) PKKKIQLHAEHALYDALMILNI XXXXXXXXXXXLEDYAFNFELILEEIARLFESG XX KDEAEKAKRMK CWMKRIKT XXXEDCQEEMANAIITILQSWIFS*

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:90, 181, and 247.

In another embodiment, the polypeptide comprises a polypeptide identicalto the amino acid sequence of SEQ ID NO:90, 181, or 247, wherein thepolypeptide (i) does not bind to human or murine IL-2Ralpha, (ii) bindsto human IL2RB with an affinity of about 11.2 nM (iii) binds to murineIL2RB with an affinity of about 16.1 nm (iv) binds to human IL-2Rβν_(c)with an affinity of about 18.8 nM and (v) binds to murine IL-2Rβν_(c)with an affinity of about 3.4 nM.

In any of these embodiments of the full length polypeptides, thepolypeptide may be an IL-4/IL-13 mimetic, wherein position 7 is I,position 8 is T or M, position 11 is E, position 14 is K, position 18 isS, position 33 is Q, position 36 is R, position 37 is F, position 39 isK, position 40 is R, position 43 is R, position 44 is N, position 46 isW, and position 47 is G. In a further embodiment, position 68 is I andposition 98 is F.

In any of these embodiments of the full length polypeptides, thepolypeptide may be an IL-2 mimetic, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or all 14 of the following are not true: position 7 isI, position 8 is T or M, position 11 is E, position 14 is K, position 18is S, position 33 is Q, position 36 is R, position 37 is F, position 39is K, position 40 is R, position 43 is R, position 44 is N, position 46is W, and position 47 is G. In a further embodiment, one or both of thefollowing are not true: position 68 is I and position 98 is F.

In one embodiment, the IL-2 mimetic polypeptides of any embodiment orcombination of embodiments disclosed herein have a three dimensionalstructure with structural coordinates having a root mean squaredeviation of common residue backbone atoms or alpha carbon atoms of lessthan 2.5 Angstroms, less than 1.5 Angstroms, or less than 1 Angstromwhen superimposed on backbone atoms or alpha carbon atoms of the threedimensional structure of native IL-2.

In another embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein have a three dimensionalstructure with structural coordinates having a root mean squaredeviation of common residue backbone atoms or alpha carbon atoms of lessthan 2.5 Angstroms, less than 1.5 Angstroms, or less than 1 Angstromwhen superimposed on backbone atoms or alpha carbon atoms of a threedimensional structure having the structural coordinates of Table E2.

In a further embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein, when in ternary complexwith the mouse IL-2 receptor βν_(c), have a three dimensional structurewherein the structural coordinates of common residue backbone atoms oralpha carbon atoms have a root mean square deviation of less than 2.5Angstroms, less than 1.5 Angstroms, or less than 1 Angstrom whensuperimposed on backbone atoms or alpha carbon atoms of the threedimensional structure of native IL-2 when in ternary complex with themouse IL-2 receptor βν_(c).

In another embodiment, the IL-4 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein have a three dimensionalstructure with structural coordinates comprising a root mean squaredeviation of common residue backbone atoms or alpha carbon atoms of lessthan 2.5 Angstroms less than 1.5 Angstroms, or less than 1 Angstrom whensuperimposed on backbone atoms or alpha carbon atoms of the threedimensional structure of native IL-4.

In each of these embodiments, the three dimensional structure of thepolypeptide may be determined using computational modeling oralternatively, the three dimensional structure of the polypeptide isdetermined using crystallographically-determined structural data.

In one embodiment of any embodiment or combination of embodimentsdisclosed herein, X1, X2, X3, and X4 are alpha-helical domains. Inanother embodiment, the amino acid length of each of X1, X2, X3 and X4is independently at least about 8, 10, 12, 14, 16, 19, or more aminoacids in length. In other embodiments, the amino acid length of each ofX1, X2, X3 and X4 is independently no more than 1000, 500, 400, 300,200, 100, or 50 amino acids in length. In various further embodiments,the amino acid length of each of X1, X2, X3 and X4 is independentlybetween about 8-1000, 8-500, 8-400, 8-300, 8-200, 8-100, 8-50, 10-1000,10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 12-1000, 12-500, 12-400,12-300, 12-200, 12-100, 12-50, 14-1000, 14-500, 14-400, 14-300, 14-200,14-100, 14-50, 16-1000, 16-500, 16-400, 16-300, 16-200, 16-100, 16-50,19-1000, 19-500, 19-400, 19-300, 19-200, 19-100, or about 19-50 aminoacids in length.

In another embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein, X1 binds to the beta andthe gamma subunit of the human IL-2 receptor. In another embodiment ofthe IL-2 mimetic polypeptides of any embodiment or combination ofembodiments disclosed herein, X2 does not bind to the human IL-2receptor. In another embodiment, of the IL-2 mimetic polypeptides of anyembodiment or combination of embodiments disclosed herein, X3 binds tothe beta subunit of the human IL-2 receptor. In a further embodiment ofthe IL-2 mimetic polypeptides of any embodiment or combination ofembodiments disclosed herein, X4 binds to the gamma subunit of the humanIL-2 receptor. In another embodiment or the IL-2 mimetic polypeptides ofany embodiment or combination of embodiments disclosed herein, thepolypeptide does not bind to the alpha subunit of the human or murineIL-2 receptor. In one embodiment, binding to the receptors is specificbinding as determined by surface plasmon resonance at biologicallyrelevant concentrations. In another embodiment, the IL-2 mimeticpolypeptides of any embodiment or combination of embodiments disclosedherein that bind to the IL-2 receptor βν_(c) heterodimer (IL-2Rβν_(c))do so with a binding affinity of 200 nm or less, 100 nm or less, 50 nMor less, or 25 nM or less. In a further embodiment of the IL-2 mimeticpolypeptides of any embodiment or combination of embodiments disclosedherein, the polypeptide's affinity for the human and mouse IL-2receptors is about equal to or greater than that of native IL-2.

In one embodiment of the IL-4 mimetic polypeptides of any embodiment orcombination of embodiments disclosed herein that bind to the IL-4receptor αν_(c) heterodimer (IL-4Rαν_(c)) do so with a binding affinityof 200 nm or less, 100 nm or less, 50 nM or less, or 25 nM or less. Inanother embodiment of the IL-4 mimetic polypeptides of any embodiment orcombination of embodiments disclosed herein, the polypeptide's affinityfor the human and mouse IL-4 receptors is about equal to or greater thanthat of native IL-4.

In one embodiment of the IL-2 mimetic polypeptides of any embodiment orcombination of embodiments disclosed herein, the polypeptide stimulatesSTAT5 phosphorylation in cells expressing the IL-2 receptor with potencyabout equal to or greater than native IL-2. In another embodiment of theIL-2 mimetic polypeptides of any embodiment or combination ofembodiments disclosed herein, the polypeptide stimulates STAT5phosphorylation in cells expressing the IL-2 receptor with potency aboutequal to or greater than native IL-2 in cells expressing IL-2 receptorβν_(c) heterodimer but lacking the IL-2 receptor α.

In another embodiment, the IL-2 mimetic polypeptides of any embodimentor combination of embodiments disclosed herein demonstrate thermalstability about equal to or greater than the thermal stability of nativeIL-2.

In a further embodiment, the polypeptides of any embodiment orcombination of embodiments disclosed herein, the polypeptides maintainor recover at least 70%, 80%, or 90% of their folded structure afterthermal stability testing, and/or maintain or recover at least 80% oftheir ellipticity spectrum after thermal stability testing, and/ormaintain or recover at least 70% or 80% of their activity after thermalstability testing. In one embodiment, such activity is determined by aSTAT5 phosphorylation assay. In another embodiment, thermal stability ismeasured by circular dichroism (CD) spectroscopy at 222 nM. In a furtherembodiment, the thermal stability test comprises heating the polypeptidefrom 25 degrees Celsius to 95 degrees Celsius in a one hour time frame,cooling the polypeptide to 25 degrees Celsius in a 5 minute time frameand monitoring ellipticity at 222 nm.

The polypeptides described herein may be chemically synthesized orrecombinantly expressed (when the polypeptide is genetically encodable).The polypeptides may be linked to other compounds, such as stabilizationcompounds to promote an increased half-life in vivo, including but notlimited to albumin, PEGylation (attachment of one or more polyethyleneglycol chains), HESylation, PASylation, glycosylation, or may beproduced as an Fc-fusion or in deimmunized variants. Such linkage can becovalent or non-covalent. For example, addition of polyethylene glycol(“PEG”) containing moieties may comprise attachment of a PEG grouplinked to maleimide group (“PEG-MAL”) to a cysteine residue of thepolypeptide. Suitable examples of PEG-MAL are methoxy PEG-MAL 5 kD;methoxy PEG-MAL 20 kD; methoxy (PEG)2-MAL 40 kD; methoxy PEG(MAL)2 5 kD;methoxy PEG(MAL)2 20 kD; methoxy PEG(MAL)2 40 kD; or any combinationthereof. See also U.S. Pat. No. 8,148,109. In other embodiments, the PEGmay comprise branched chain PEGs and/or multiple PEG chains.

In one embodiment, the stabilization compound, including but not limitedto a PEG-containing moiety, is linked at a cysteine residue in thepolypeptide. In another embodiment, the cysteine residue is present inthe X2 domain. In some embodiments, the cysteine residue is present, forexample, in any one of a number of positions in the X2 domain. In somesuch embodiments, the X2 domain is at least 19 amino acids in length andthe cysteine residue is at positions 1, 2, 5, 9 or 16 relative to those19 amino acids. In a further embodiment, the stabilization compound,including but not limited to a PEG-containing moiety, is linked to thecysteine residue via a maleimide group, including but not limited tolinked to a cysteine residue present at amino acid residue 62 relativeto SEQ ID NO:90.

In some aspects, the polypeptide is a Neo-2/15 polypeptide and an aminoacid of Neo-2/15 is mutated to a cysteine residue for attachment of astabilization moiety (e.g., PEG-containing moiety) thereto. In someaspects, the polypeptide is a Neo-2/15 polypeptide and the amino acid atpositions 50, 53, 62, 69, 73, 82, 56, 58, 59, 66, 77, or 85 or acombination thereof relative to SEQ ID NO:90, 181, or 247 is mutated toa cysteine residue for attachment of a stabilization moiety (e.g.,PEG-containing moiety) thereto. Accordingly, in a further embodiment,the polypeptide comprises a polypeptide at least 25%, 27%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%identical to the full length of the amino acid sequence of SEQ ID NO:90,181, or 247 [Neo-2/15], and wherein one, two, three, four, five, or allsix of the following mutations are present:

R50C;

E53C;

E62C;

E69C;

R73C; and/or

E82C.

In a further embodiment, the polypeptide comprises a polypeptide atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical to the full length of the aminoacid sequence of SEQ ID NO:90, 181, or 247, and wherein one, two, three,four, five, six, seven, eight, nine, ten, eleven, or all twelve of thefollowing mutations are present

D56C;

K58C;

D59C;

R66C;

T77C;

E85C;

R50C;

E53C;

E62C;

E69C;

R73C; and/or

E82C.

In a further embodiment, the polypeptide comprises a polypeptide atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical to the full length of the aminoacid sequence selected from the group consisting of SEQ ID NOS: 190-243.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:190 and 217. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:190.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:191 and 218. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:191.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:192 and 219. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:192.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:193 and 220. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:193.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:194 and 221. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:194.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:195 and 222. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:195.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:196 and 223. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:196.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:197 and 224. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:197.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:198 and 225. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:198.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:199 and 226. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:199.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:200 and 227. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:200.

In one embodiment, the polypeptide comprises a polypeptide at least atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalalong its length to the amino acid sequence selected from the groupconsisting of SEQ ID NO:201 and 228. In one aspect, the polypeptidecomprises a polypeptide at least at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical along its length to the amino acidsequence of SEQ ID NO:201.

In another embodiment, the polypeptide comprises a polypeptide at least25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 100% identical to the full length of the amino acidsequence selected from the group consisting of SEQ ID NO:195, 207, 214,222, 234, and 241; or wherein the polypeptide comprises a polypeptide atleast 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 100% identical to the full length of the aminoacid sequence selected from the group consisting of SEQ ID NO:195, 207,and 214.

In a further embodiment, the polypeptide further comprises a targetingdomain. In this embodiment, the polypeptide can be directed to a targetof interest. The targeting domain may be covalently or non-covalentlybound to the polypeptide. In embodiments where the targeting domain isnon-covalently bound to the polypeptide, any suitable means for suchnon-covalent binding may be used, including but not limited tostreptavidin-biotin linkers.

In another embodiment, the targeting domain, when present, is atranslational fusion with the polypeptide. In this embodiment, thepolypeptide and the targeting domain may directly abut each other in thetranslational fusion or may be linked by a polypeptide linker suitablefor an intended purpose. Exemplary such linkers include, but are notlimited, to those disclosed in WO2016178905, WO2018153865 (inparticular, at page 13), and WO 2018170179 (in particular, at paragraphs[0316]40317D. In other embodiments, suitable linkers include, but arenot limited to peptide linkers, such as GGGGG (SEQ ID NO: 95), GSGGG(SEQ ID NO: 96), GGGGGG (SEQ ID NO: 97), GGSGGG (SEQ ID NO: 98),GGSGGSGGGSGGSGSG (SEQ ID NO: 99), GSGGSGGGSGGSGSG (SEQ ID NO: 100),GGSGGSGGGSGGSGGGGSGGSGGGSGGGGS (SEQ ID NO: 101), and [GGGGX]_(n) (SEQ IDNO: 102), where X is Q, E or S and n is 2-5.

The targeting domains are polypeptide domains or small molecules thatbind to a target of interest. In one non-limiting embodiment, thetargeting domain binds to a cell surface protein; in this embodiment,the cell may be any cell type of interest that includes a surfaceprotein that can be bound by a suitable targeting domain. In oneembodiment, the cell surface proteins are present on the surface ofcells selected from the group consisting of tumor cells, tumor vascularcomponent cells, tumor microenvironment cells (e.g. fibroblasts,infiltrating immune cells, or stromal elements), other cancer cells andimmune cells (including but not limited to CD8+ T cells, T-regulatorycells, dendritic cells, NK cells, or macrophages). When the cell surfaceprotein is on the surface of a tumor cell, vascular component cell, ortumor microenvironment cell (e.g. fibroblasts, infiltrating immunecells, or stromal elements), any suitable tumor cell, vascular componentcell, or tumor microenvironment cell surface marker may be targeted,including but not limited to EGFR, EGFRvIII, Her2, HER3, EpCAM, MSLN,MUC16, PSMA, TROP2, ROR1, RON, PD-L1, CD47, CTLA-4, CD5, CD19, CD20,CD25, CD37, CD30, CD33, CD40, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3,B7-DC, HLD-DR, carcinoembryonic antigen (CEA), TAG-72, MUC1,folate-binding protein, A33, G250, prostate-specific membrane antigen(PSMA), ferritin, GD2, GD3, GM2, Le^(y), CA-125, CA19-9, epidermalgrowth factor, p185HER2, IL-2 receptor, EGFRvIII (del-7 EGFR),fibroblast activation protein, tenascin, a metalloproteinase,endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6,HPV E7, Her-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, MelanA/MART1, Rasmutant, gp100, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA,hTERT, a Sarcoma translocation breakpoint protein, EphA2, PAP, ML-IAP,AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B 1, polysialicacid, MYCN, RhoC, TRP-2, fucosy1 GM1, mesothelin (MSLN), PSCA, MAGE A1,sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1,RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TESL Sperm protein 17,LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, Legumain, Tie 3, VEGFR2, MAD-CT-1,PDGFR-B, MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR,CMET, HER3, MUC15, CA6, NAPI2B, TROP2, CLDN6, CLDN16, CLDN18.2,CLorf186, RON, LY6E, FRA, DLL3, PTK7, STRA6, TMPRSS3, TMPRSS4, TMEM238,UPK1B, VTCN1, LIV1, ROR1, and Fos-related antigen 1.

In other embodiments, when the cell surface protein is on the surface ofa tumor cell, vascular component cell, or tumor microenvironment cell(e.g. fibroblasts, infiltrating immune cells, or stromal elements), anysuitable tumor cell, vascular component cell, or tumor microenvironmentcell surface marker may be targeted, including but not limited totargets in the following list:

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM. sub.-001203);(2) E16 (LAT1, SLC7A5, Genbank accession no. NM.sub.-003486);(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM.sub.-012449);(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM.sub.-005823);(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM.sub.-006424);(7) Sema 5b (FLJ10372, KIAA1445, Mm. 42015, SEMASB, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878);(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628);(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);(10) MSG783 (RNF124, hypothetical protein F1120315, Genbank accessionno. NM.sub.-017763);(11) STEAP2 (HGNC.sub.-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP,prostate cancer associated gene 1, prostate cancer associated protein 1,six transmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138);(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM.sub.-017636);(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP.sub.-003203 or NM.sub.-003212);(14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr virusreceptor) or Hs. 73792, Genbank accession no. M26004);(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank accessionno. NM.sub.-000626);(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_-030764);(17) HER2 (Genbank accession no. M11730);(18) NCA (Genbank accession no. M18728);(19) MDP (Genbank accession no. BC017023);(20) IL20R.alpha. (Genbank accession no. AF184971);(21) Brevican (Genbank accession no. AF229053);(22) Ephb2R (Genbank accession no. NM_-004442);(23) ASLG659 (Genbank accession no. AX092328);(24) PSCA (Genbank accession no. AJ297436);(25) GEDA (Genbank accession no. AY260763);(26) BAFF-R (Genbank accession no. NP_-443177.1);(27) CD22 (Genbank accession no. NP-001762.1);(28) CD79a (CD79A, CD79.alpha., immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation, Genbank accession No. NP_-001774.1);

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbankaccession No. NP_-001707.1);

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+ T lymphocytes, Genbankaccession No. NP_-002111.1);(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability, Genbank accessionNo. NP_-002552.2);(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accessionNo. NP_-001773.1);(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis, Genbankaccession No. NP_-005573.1);(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation, Genbank accession No.NP_-443170.1); or(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies, Genbank accession No. NP_-112571.1).

In another embodiment, the targeting domain binds to immune cell surfacemarkers. In this embodiment, the target may be cell surface proteins onany suitable immune cell, including but not limited to CD8+ T cells,T-regulatory cells, dendritic cells, NK cells or macrophages. Thetargeting domain may target any suitable immune cell surface marker(whether an endogenous or an engineered immune cell, including but notlimited to engineered CAR-T cells), including but not limited to CD3,CD4, CD8, CD19, CD20, CD21, CD25, CD37, CD30, CD33, CD40, CD68, CD123,CD254, PD-1, B7-H3, and CTLA-4. In another embodiment, the targetingdomain binds to PD-1, PDL-1, CTLA-4, TROP2, B7-H3, CD33, CD22, carbonicanhydrase IX, CD123, Nectin-4, tissue factor antigen, CD154, B7-H3,B7-H4, FAP (fibroblast activation protein) or MUC16, and/or wherein thetargeting domain binds to PD-1, PDL-1, CTLA-4, TROP2, B7-H3, CD33, CD22,carbonic anhydrase IX, CD123, Nectin-4, tissue factor antigen, CD154,B7-H3, B7-H4, FAP (fibroblast activation protein) or MUC16.

In all these embodiments, the targeting domains can be any suitablepolypeptides that bind to targets of interest and can be incorporatedinto the polypeptide of the disclosure. In non-limiting embodiments, thetargeting domain may include but is not limited to an scFv, a F(ab), aF(ab′)₂, a B cell receptor (BCR), a DARPin, an affibody, a monobody, ananobody, diabody, an antibody (including a monospecific or bispecificantibody); a cell-targeting oligopeptide including but not limited toRGD integrin-binding peptides, de novo designed binders, aptamers, abicycle peptide, conotoxins, small molecules such as folic acid, and avirus that binds to the cell surface.

In another embodiment, the polypeptides include at least one disulfidebond (i.e.: 1, 2, 3, 4, or more disulfide bonds). Any suitable disulfidebonds may be used, such as disulfide bonds linking two differenthelices. In one embodiment, the disulfide bonds include a disulfide bondlinking helix 1 (X1) and helix 4 (X4). The disulfide bond may, forexample, improve the thermal stability of the polypeptide as compared toa substantially similar polypeptide with no disulfide bond linking twodomains together.

The polypeptides and peptide domains of the invention may includeadditional residues at the N-terminus, C-terminus, or both that are notpresent in the polypeptides or peptide domains of the disclosure; theseadditional residues are not included in determining the percent identityof the polypeptides or peptide domains of the disclosure relative to thereference polypeptide. Such residues may be any residues suitable for anintended use, including but not limited to detection tags (i.e.:fluorescent proteins, antibody epitope tags, etc.), adaptors, ligandssuitable for purposes of purification (His tags, etc.), other peptidedomains that add functionality to the polypeptides, etc. Residuessuitable for attachment of such groups may include cysteine, lysine orp-acetylphenylalanine residues or can be tags, such as amino acid tagssuitable for reaction with transglutaminases as disclosed in U.S. Pat.Nos. 9,676,871 and 9,777,070.

In a further aspect, the present invention provides nucleic acids,including isolated nucleic acids, encoding a polypeptide of the presentinvention that can be genetically encoded. The isolated nucleic acidsequence may comprise RNA or DNA. Such isolated nucleic acid sequencesmay comprise additional sequences useful for promoting expression and/orpurification of the encoded protein, including but not limited to polyAsequences, modified Kozak sequences, and sequences encoding epitopetags, export signals, and secretory signals, nuclear localizationsignals, and plasma membrane localization signals. It will be apparentto those of skill in the art, based on the teachings herein, whatnucleic acid sequences will encode the polypeptides of the invention.

In another aspect, the present invention provides recombinant expressionvectors comprising the isolated nucleic acid of any aspect of theinvention operatively linked to a suitable control sequence.“Recombinant expression vector” includes vectors that operatively link anucleic acid coding region or gene to any control sequences capable ofeffecting expression of the gene product. “Control sequences” operablylinked to the nucleic acid sequences of the invention are nucleic acidsequences capable of effecting the expression of the nucleic acidmolecules. The control sequences need not be contiguous with the nucleicacid sequences, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the nucleicacid sequences and the promoter sequence can still be considered“operably linked” to the coding sequence. Other such control sequencesinclude, but are not limited to, polyadenylation signals, terminationsignals, and ribosome binding sites. Such expression vectors include butare not limited to, plasmid and viral-based expression vectors. Thecontrol sequence used to drive expression of the disclosed nucleic acidsequences in a mammalian system may be constitutive (driven by any of avariety of promoters, including but not limited to, CMV, SV40, RSV,actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive). The expression vector must be replicable in thehost organisms either as an episome or by integration into hostchromosomal DNA. In various embodiments, the expression vector maycomprise a plasmid, viral-based vector (including but not limited to aretroviral vector or oncolytic virus), or any other suitable expressionvector. In some embodiments, the expression vector can be administeredin the methods of the disclosure to express the polypeptides in vivo fortherapeutic benefit. In non-limiting embodiments, the expression vectorscan be used to transfect or transduce cell therapeutic targets(including but not limited to CAR-T cells or tumor cells) to effect thetherapeutic methods disclosed herein.

In a further aspect, the present disclosure provides host cells thatcomprise the recombinant expression vectors disclosed herein, whereinthe host cells can be either prokaryotic or eukaryotic. The cells can betransiently or stably engineered to incorporate the expression vector ofthe invention, using techniques including but not limited to bacterialtransformations, calcium phosphate co-precipitation, electroporation, orliposome mediated-, DEAE dextran mediated-, polycationic mediated-, orviral mediated transfection. (See, for example, Molecular Cloning: ALaboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor LaboratoryPress); Culture of Animal Cells: A Manual of Basic Technique, 2^(nd) Ed.(R. I. Freshney. 1987. Liss, Inc. New York, N.Y.)). A method ofproducing a polypeptide according to the invention is an additional partof the invention. The method comprises the steps of (a) culturing a hostaccording to this aspect of the invention under conditions conducive tothe expression of the polypeptide, and (b) optionally, recovering theexpressed polypeptide. The expressed polypeptide can be recovered fromthe cell free extract, but preferably they are recovered from theculture medium.

In a further aspect, the present disclosure provides antibodies thatselectively bind to the polypeptides of the disclosure. The antibodiescan be polyclonal, monoclonal antibodies, humanized antibodies, andfragments thereof, and can be made using techniques known to those ofskill in the art. As used herein, “selectively bind” means preferentialbinding of the antibody to the polypeptide of the disclosure, as opposedto one or more other biological molecules, structures, cells, tissues,etc., as is well understood by those of skill in the art.

In another aspect, the present disclosure provides pharmaceuticalcompositions, comprising one or more polypeptides, nucleic acids,expression vectors, and/or host cells of the disclosure and apharmaceutically acceptable carrier. The pharmaceutical compositions ofthe disclosure can be used, for example, in the methods of thedisclosure described below. The pharmaceutical composition may comprisein addition to the polypeptide of the disclosure (a) a lyoprotectant;(b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent;(e) a stabilizer; (f) a preservative and/or (g) a buffer.

In some embodiments, the buffer in the pharmaceutical composition is aTris buffer, a histidine buffer, a phosphate buffer, a citrate buffer oran acetate buffer. The pharmaceutical composition may also include alyoprotectant, e.g. sucrose, sorbitol or trehalose. In certainembodiments, the pharmaceutical composition includes a preservative e.g.benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol,benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol,p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoicacid, and various mixtures thereof. In other embodiments, thepharmaceutical composition includes a bulking agent, like glycine. Inyet other embodiments, the pharmaceutical composition includes asurfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. The pharmaceutical composition mayalso include a tonicity adjusting agent, e.g., a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes a stabilizer, e.g., a molecule which,when combined with a protein of interest substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized or liquid form. Exemplary stabilizers include sucrose,sorbitol, glycine, inositol, sodium chloride, methionine, arginine, andarginine hydrochloride.

The polypeptides, nucleic acids, expression vectors, and/or host cellsmay be the sole active agent in the pharmaceutical composition, or thecomposition may further comprise one or more other active agentssuitable for an intended use.

In a further aspect, the present disclosure provides methods fortreating and/or limiting cancer, comprising administering to a subjectin need thereof a therapeutically effective amount of one or morepolypeptides, nucleic acids, expression vectors, and/or host cells ofthe disclosure, salts thereof, conjugates thereof, or pharmaceuticalcompositions thereof, to treat and/or limit the cancer. When the methodcomprises treating cancer, the one or more polypeptides, nucleic acids,expression vectors, and/or host cells are administered to a subject thathas already been diagnosed as having cancer. As used herein, “treat” or“treating” means accomplishing one or more of the following: (a)reducing the size or volume of tumors and/or metastases in the subject;(b) limiting any increase in the size or volume of tumors and/ormetastases in the subject; (c) increasing survival; (d) reducing theseverity of symptoms associated with cancer; (e) limiting or preventingdevelopment of symptoms associated with cancer; and (f) inhibitingworsening of symptoms associated with cancer.

When the method comprises limiting development of cancer, the one ormore polypeptides, nucleic acids, expression vectors, and/or host cellsare administered prophylactically to a subject that is not known to havecancer, but may be at risk of cancer. As used herein, “limiting” meansto limit development of cancer in subjects at risk of cancer, includingbut not limited to subjects with a family history of cancer, subjectsgenetically predisposed to cancer, subjects that are symptomatic forcancer, etc.

The methods can be used to treat or limit development of any suitablecancer, including but not limited to colon cancer, melanoma, renal cellcancer, head and neck squamous cell cancer, gastric cancer, urothelialcarcinoma, Hodgkin lymphoma, non-small cell lung cancer, small cell lungcancer, hepatocellular carcinoma, pancreatic cancer, Merkel cellcarcinoma colorectal cancer, acute myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiplemyeloma, ovarian cancer, cervical cancer, and any tumor types selectedby a diagnostic test, such as microsatellite instability, tumormutational burden, PD-L1 expression level, or the immunoscore assay (asdeveloped by the Society for Immunotherapy of Cancer).

The subject may be any subject that has or is at risk of developingcancer. In one embodiment, the subject is a mammal, including but notlimited to humans, dogs, cats, horses, cattle, etc.

In a further aspect, the present disclosure provides methods formodulating an immune response in a subject by administering to a subjecta polypeptide, recombinant nucleic acid, expression vector, recombinanthost cell, or the pharmaceutical composition of the present disclosure.

As used herein, an “immune response” being modulated refers to aresponse by a cell of the immune system, such as a B cell, T cell (CD4or CD8), regulatory T cell, antigen-presenting cell, dendritic cell,monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, orneutrophil, to a stimulus. In some embodiments, the response is specificfor a particular antigen (an “antigen-specific response”), and refers toa response by a CD4 T cell, CD8 T cell, or B cell via theirantigen-specific receptor. In some embodiments, an immune response is aT cell response, such as a CD4+ response or a CD8+ response. Suchresponses by these cells can include, for example, cytotoxicity,proliferation, cytokine or chemokine production, trafficking, orphagocytosis, and can be dependent on the nature of the immune cellundergoing the response. In some embodiments of the compositions andmethods described herein, an immune response being modulated is T-cellmediated.

In some aspects, the immune response is an anti-cancer immune response.In some such aspects, an IL-2 mimetic described herein is administeredto a subject having cancer to modulate an anti-cancer immune response inthe subject.

In some aspects, the immune response is a tissue reparative immuneresponse. In some such aspects, an IL-4 mimetic described here isadministered to a subject in need thereof to modulate a tissuereparative immune response in the subject.

In some aspects, the immune response is a wound healing immune response.In some such aspects, an IL-4 mimetic described here is administered toa subject in need thereof to modulate a wound healing immune response inthe subject.

In some aspects, methods are provided for modulating an immune responseto a second therapeutic agent in a subject. In some such aspects, themethod comprises administering a polypeptide of the present disclosurein combination with an effective amount of the second therapeutic agentto the subject. The second therapeutic agent can be, for example, achemotherapeutic agent or an antigen-specific immunotherapeutic agent.In some aspects, the antigen-specific immunotherapeutic agent compriseschimeric antigen receptor T cells (CAR-T cells). In some aspects, thepolypeptide of the present disclosure enhances the immune response ofthe subject to the therapeutic agent. The immune response can beenhanced, for example, by improving the T cell response (including CAR-Tcell response), augmenting the innate T cell immune response, decreasinginflammation, inhibiting T regulatory cell activity, or combinationsthereof.

In some aspects, a cytokine mimetic of the present invention, e.g., anIL-4 mimetic as described herein, will be impregnated to or otherwiseassociated with a biomaterial and the biomaterial will be introduced toa subject. In some aspects, the biomaterial will be a component of animplantable medical device and the device will be, for example, coatedwith the biomaterial. Such medical devices include, for example,vascular and arterial grafts. IL-4 and/or IL-4 associated biomaterialscan be used, for example, to promote wound healing and/or tissue repairand regeneration.

As used herein, a “therapeutically effective amount” refers to an amountof the polypeptide, nucleic acids, expression vectors, and/or host cellsthat is effective for treating and/or limiting cancer. The polypeptides,nucleic acids, expression vectors, and/or host cells are typicallyformulated as a pharmaceutical composition, such as those disclosedabove, and can be administered via any suitable route, including but notlimited to orally, by inhalation spray, ocularly, intravenously,subcutaneously, intraperitoneally, and intravesicularly in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. In one particular embodiment, thepolypeptides, nucleic acids, expression vectors, and/or host cells areadministered mucosally, including but not limited to intraocular,inhaled, or intranasal administration. In another particular embodiment,the polypeptides, nucleic acids, expression vectors, and/or host cellsare administered orally. Such particular embodiments can be administeredvia droplets, nebulizers, sprays, or other suitable formulations.

Any suitable dosage range may be used as determined by attending medicalpersonnel. Dosage regimens can be adjusted to provide the optimumdesired response (e.g., a therapeutic or prophylactic response). Asuitable dosage range for the polypeptides may, for instance, be 0.1ug/kg-100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to 50mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight. In someembodiments, the recommended dose could be lower than 0.1 mcg/kg,especially if administered locally. In other embodiments, therecommended dose could be based on weight/m² (i.e. body surface area),and/or it could be administered at a fixed dose (e.g., 0.05-100 mg). Thepolypeptides, nucleic acids, expression vectors, and/or host cells canbe delivered in a single bolus, or may be administered more than once(e.g., 2, 3, 4, 5, or more times) as determined by an attendingphysician.

The polypeptides, nucleic acids, expression vectors, and/or host cellsmade be administered as the sole prophylactic or therapeutic agent, ormay be administered together with (i.e.: combined or separately) one ormore other prophylactic or therapeutic agents, including but not limitedto tumor resection, chemotherapy, radiation therapy, immunotherapy, etc.

Example Computing Environment

FIG. 22 is a block diagram of an example computing network. Some or allof the above-mentioned techniques disclosed herein, such as but notlimited to techniques disclosed as part of and/or being performed bysoftware, the Rosetta software suite, RosettaScripts, PyRosetta, Rosettaapplications, and/or other herein-described computer software andcomputer hardware, can be part of and/or performed by a computingdevice. For example, FIG. X1 shows protein design system 102 configuredto communicate, via network 106, with client devices 104 a, 104 b, and104 c and protein database 108. In some embodiments, protein designsystem 102 and/or protein database 108 can be a computing deviceconfigured to perform some or all of the herein described methods andtechniques, such as but not limited to, method 300 and functionalitydescribed as being part of or related to Rosetta. Protein database 108can, in some embodiments, store information related to and/or used byRosetta.

Network 106 may correspond to a LAN, a wide area network (WAN), acorporate intranet, the public Internet, or any other type of networkconfigured to provide a communications path between networked computingdevices. Network 106 may also correspond to a combination of one or moreLANs, WANs, corporate intranets, and/or the public Internet.

Although FIG. 22 only shows three client devices 104 a, 104 b, 104 c,distributed application architectures may serve tens, hundreds, orthousands of client devices. Moreover, client devices 104 a, 104 b, 104c (or any additional client devices) may be any sort of computingdevice, such as an ordinary laptop computer, desktop computer, networkterminal, wireless communication device (e.g., a cell phone or smartphone), and so on. In some embodiments, client devices 104 a, 104 b, 104c can be dedicated to problem solving/using the Rosetta software suite.In other embodiments, client devices 104 a, 104 b, 104 c can be used asgeneral purpose computers that are configured to perform a number oftasks and need not be dedicated to problem solving/using the Rosettasoftware suite. In still other embodiments, part or all of thefunctionality of protein design system 102 and/or protein database 108can be incorporated in a client device, such as client device 104 a, 104b, and/or 104 c.

Computing Environment Architecture

FIG. 23A is a block diagram of an example computing device (e.g.,system) In particular, computing device 200 shown in FIG. 23A can beconfigured to: include components of and/or perform one or morefunctions of some or all of the herein described methods and techniques,such as but not limited to, method 300 and functionality described asbeing part of or related to Rosetta. Computing device 200 may include auser interface module 201, a network-communication interface module 202,one or more processors 203, data storage 204, and protein synthesisdevice 220, all of which may be linked together via a system bus,network, or other connection mechanism 205.

User interface module 201 can be operable to send data to and/or receivedata from external user input/output devices. For example, userinterface module 201 can be configured to send and/or receive data toand/or from user input devices such as a keyboard, a keypad, a touchscreen, a computer mouse, a track ball, a joystick, a camera, a voicerecognition module, and/or other similar devices. User interface module201 can also be configured to provide output to user display devices,such as one or more cathode ray tubes (CRT), liquid crystal displays(LCD), light emitting diodes (LEDs), displays using digital lightprocessing (DLP) technology, printers, light bulbs, and/or other similardevices, either now known or later developed. User interface module 201can also be configured to generate audible output(s), such as a speaker,speaker jack, audio output port, audio output device, earphones, and/orother similar devices.

Network-communications interface module 202 can include one or morewireless interfaces 207 and/or one or more wireline interfaces 208 thatare configurable to communicate via a network, such as network 106 shownin FIG. 22. Wireless interfaces 207 can include one or more wirelesstransmitters, receivers, and/or transceivers, such as a Bluetoothtransceiver, a Zigbee transceiver, a Wi-Fi transceiver, a WiMAXtransceiver, and/or other similar type of wireless transceiverconfigurable to communicate via a wireless network. Wireline interfaces208 can include one or more wireline transmitters, receivers, and/ortransceivers, such as an Ethernet transceiver, a Universal Serial Bus(USB) transceiver, or similar transceiver configurable to communicatevia a twisted pair, one or more wires, a coaxial cable, a fiber-opticlink, or a similar physical connection to a wireline network.

In some embodiments, network communications interface module 202 can beconfigured to provide reliable, secured, and/or authenticatedcommunications. For each communication described herein, information forensuring reliable communications (i.e., guaranteed message delivery) canbe provided, perhaps as part of a message header and/or footer (e.g.,packet/message sequencing information, encapsulation header(s) and/orfooter(s), size/time information, and transmission verificationinformation such as CRC and/or parity check values). Communications canbe made secure (e.g., be encoded or encrypted) and/or decrypted/decodedusing one or more cryptographic protocols and/or algorithms, such as,but not limited to, DES, AES, RSA, Diffie-Hellman, and/or DSA. Othercryptographic protocols and/or algorithms can be used as well or inaddition to those listed herein to secure (and then decrypt/decode)communications.

Processors 203 can include one or more general purpose processors and/orone or more special purpose processors (e.g., digital signal processors,application specific integrated circuits, etc.). Processors 203 can beconfigured to execute computer-readable program instructions 206contained in data storage 204 and/or other instructions as describedherein. Data storage 204 can include one or more computer-readablestorage media that can be read and/or accessed by at least one ofprocessors 203. The one or more computer-readable storage media caninclude volatile and/or non-volatile storage components, such asoptical, magnetic, organic or other memory or disc storage, which can beintegrated in whole or in part with at least one of processors 203. Insome embodiments, data storage 204 can be implemented using a singlephysical device (e.g., one optical, magnetic, organic or other memory ordisc storage unit), while in other embodiments, data storage 204 can beimplemented using two or more physical devices.

Data storage 204 can include computer-readable program instructions 206and perhaps additional data. For example, in some embodiments, datastorage 204 can store part or all of data utilized by a protein designsystem and/or a protein database; e.g., protein designs system 102,protein database 108. In some embodiments, data storage 204 canadditionally include storage required to perform at least part of theherein-described methods and techniques and/or at least part of thefunctionality of the herein-described devices and networks.

In some examples, computing device 200 includes protein synthesis device220. Protein synthesis device can synthesize (or generate polypeptidesbased on input data provided to protein synthesis device 220 usingcommands and/or data provided by processors 203 and/or data storage 204.For example, part or all of the functionality of protein synthesisdevice 220 can be performed by a semi-automated or an automated peptidesynthesizer.

FIG. 23B depicts a network 106 of computing clusters 209 a, 209 b, 209 carranged as a cloud-based server system in accordance with an exampleembodiment. Data and/or software for protein design system 102 can bestored on one or more cloud-based devices that store program logicand/or data of cloud-based applications and/or services. In someexamples, protein design system 102 can be a single computing deviceresiding in a single computing center. In other examples, protein designsystem 102 can include multiple computing devices in a single computingcenter, or even multiple computing devices located in multiple computingcenters located in diverse geographic locations.

In some examples, data and/or software for protein design system 102 canbe encoded as computer readable information stored in tangible computerreadable media (or computer readable storage media) and accessible byclient devices 104 a, 104 b, and 104 c, and/or other computing devices.In some examples, data and/or software for protein design system 102 canbe stored on a single disk drive or other tangible storage media, or canbe implemented on multiple disk drives or other tangible storage medialocated at one or more diverse geographic locations.

FIG. 23B depicts a cloud-based server system in accordance with anexample embodiment. In FIG. 23B, the functions of protein design system102 can be distributed among three computing clusters 209 a, 209 b, and209 c. Computing cluster 209 a can include one or more computing devices200 a, cluster storage arrays 210 a, and cluster routers 211 a connectedby a local cluster network 212 a. Similarly, computing cluster 209 b caninclude one or more computing devices 200 b, cluster storage arrays 210b, and cluster routers 211 b connected by a local cluster network 212 b.Likewise, computing cluster 209 c can include one or more computingdevices 200 c, cluster storage arrays 210 c, and cluster routers 211 cconnected by a local cluster network 212 c.

In some examples, each of the computing clusters 209 a, 209 b, and 209 ccan have an equal number of computing devices, an equal number ofcluster storage arrays, and an equal number of cluster routers. In otherexamples, however, each computing cluster can have different numbers ofcomputing devices, different numbers of cluster storage arrays, anddifferent numbers of cluster routers. The number of computing devices,cluster storage arrays, and cluster routers in each computing clustercan depend on the computing task or tasks assigned to each computingcluster.

In computing cluster 209 a, for example, computing devices 200 a can beconfigured to perform various computing tasks of protein design system102. In one example, the various functionalities of protein designsystem 102 can be distributed among one or more of computing devices 200a, 200 b, and 200 c. Computing devices 200 b and 200 c in computingclusters 209 b and 209 c can be configured similarly to computingdevices 200 a in computing cluster 209 a. On the other hand, in someexamples, computing devices 200 a, 200 b, and 200 c can be configured toperform different functions.

In some examples, computing tasks and stored data associated withprotein design system 102 can be distributed across computing devices200 a, 200 b, and 200 c based at least in part on the processingrequirements of protein design system 102, the processing capabilitiesof computing devices 200 a, 200 b, and 200 c, the latency of the networklinks between the computing devices in each computing cluster andbetween the computing clusters themselves, and/or other factors that cancontribute to the cost, speed, fault-tolerance, resiliency, efficiency,and/or other design goals of the overall system architecture.

The cluster storage arrays 210 a, 210 b, and 210 c of the computingclusters 209 a, 209 b, and 209 c can be data storage arrays that includedisk array controllers configured to manage read and write access togroups of hard disk drives. The disk array controllers, alone or inconjunction with their respective computing devices, can also beconfigured to manage backup or redundant copies of the data stored inthe cluster storage arrays to protect against disk drive or othercluster storage array failures and/or network failures that prevent oneor more computing devices from accessing one or more cluster storagearrays.

Similar to the manner in which the functions of protein design system102 can be distributed across computing devices 200 a, 200 b, and 200 cof computing clusters 209 a, 209 b, and 209 c, various active portionsand/or backup portions of these components can be distributed acrosscluster storage arrays 210 a, 210 b, and 210 c. For example, somecluster storage arrays can be configured to store one portion of thedata and/or software of protein design system 102, while other clusterstorage arrays can store a separate portion of the data and/or softwareof protein design system 102. Additionally, some cluster storage arrayscan be configured to store backup versions of data stored in othercluster storage arrays.

The cluster routers 211 a, 211 b, and 211 c in computing clusters 209 a,209 b, and 209 c can include networking equipment configured to provideinternal and external communications for the computing clusters. Forexample, the cluster routers 211 a in computing cluster 209 a caninclude one or more internet switching and routing devices configured toprovide (i) local area network communications between the computingdevices 200 a and the cluster storage arrays 201 a via the local clusternetwork 212 a, and (ii) wide area network communications between thecomputing cluster 209 a and the computing clusters 209 b and 209 c viathe wide area network connection 213 a to network 106. Cluster routers211 b and 211 c can include network equipment similar to the clusterrouters 211 a, and cluster routers 211 b and 211 c can perform similarnetworking functions for computing clusters 209 b and 209 b that clusterrouters 211 a perform for computing cluster 209 a.

In some examples, the configuration of the cluster routers 211 a, 211 b,and 211 c can be based at least in part on the data communicationrequirements of the computing devices and cluster storage arrays, thedata communications capabilities of the network equipment in the clusterrouters 211 a, 211 b, and 211 c, the latency and throughput of localnetworks 212 a, 212 b, 212 c, the latency, throughput, and cost of widearea network links 213 a, 213 b, and 213 c, and/or other factors thatcan contribute to the cost, speed, fault-tolerance, resiliency,efficiency and/or other design goals of the moderation systemarchitecture.

Example Methods of Operation

FIG. 24 is a flow chart of an example method 300. Method 300 can becarried out by a computing device, such as computing device 200described in the context of at least FIG. 2A. At least the examples ofmethod 300 mentioned below are discussed above.

Method 300 can begin at block 310, where the computing device candetermine a structure for a plurality of residues of a protein using acomputing device, where the structure of the plurality of residuesprovides a particular receptor binding interface. As will be understoodby the skilled practitioner, the determining of a structure for aplurality of residues of a protein where the structure of the pluralityof residues provides a particular receptor binding interface istypically the identification of the original residues of a nativeprotein that bind to a particular receptor binding interface whereas theplurality of designed residues are identified residues that can bind tothe same receptor binding interface.

At block 320, the computing device can determine a plurality of designedresidues using a mimetic design protocol, where the plurality ofdesigned residues provide the particular receptor binding interface, andwhere the plurality of designed residues differ from the plurality ofresidues.

In some examples, determining the plurality of designed residues usingthe mimetic design protocol can include determining an idealized residueusing a database of idealized residues, where the idealized residue isrelated to a designed residue of the plurality of designed residues. Insome of these examples, determining the idealized residue using thedatabase of idealized residues can include: retrieving one or moreidealized fragments related to the idealized residue from the databaseof idealized residues; and determining the idealized residue byreconstructing the related designed residue using the one or moreidealized fragments. In some of these examples, reconstructing therelated designed residue using the one or more idealized fragments caninclude: reconnecting pairs of the one or more idealized fragments by:use of combinatorial fragment assembly of the pairs of the one or moreidealized fragments; and using Cartesian-constrained backboneminimization to determine whether the pairs of the one or more idealizedfragments link two or more of the plurality of designed residues. Insome of these examples, reconstructing the related designed residueusing the one or more idealized fragments can include: verifying thatoverlapping fragments of the idealized residue are idealized fragmentsusing the database of idealized residues; verifying whether theidealized residue does not clash with a target receptor associated withthe particular receptor binding interface; and after verifying that theidealized residue does not clash with a target receptor associated withthe particular receptor binding interface, determining a most probableamino acid at each position of the idealized residue using the databaseof idealized residues. In some of these examples, determining the firstprotein backbone for the protein by assembling the one or moreconnecting helix structures and the plurality of designed residues overthe plurality of combinations can include: recombining the pairs of theone or more idealized fragments by combinatorially recombining the pairsof the one or more idealized fragments; and determining the firstprotein backbone for the protein using the recombined pairs of the oneor more idealized fragments. In some of these examples, combinatoriallyrecombining the pairs of the one or more idealized fragments can includeranking the pairs of the one or more idealized fragments based on aninterconnection length between idealized fragments of the pairs of theone or more idealized fragments.

In other examples, determining the plurality of designed residues usingthe mimetic design protocol can include: determining an idealizedresidue using one or more parametric equations that represent a shape ofa designed residue of the plurality of designed residues; anddetermining a single fragment that closes the idealized residue with atleast one designed residue of the plurality of designed residues. Insome of these examples, the designed residue can include a helicalstructure, and the one or more parametric equations can include anequation related to phi and psi angles of the helical structure. In someof these examples, the equation related to phi and psi angles of thehelical structure can include one or more terms related to an angularpitch of the phi and psi angles of the helical structure.

At block 330, the computing device can determine one or more connectinghelix structures that connect the plurality of designed residues.

At block 340, the computing device can determine a first proteinbackbone for the protein by assembling the one or more connecting helixstructures and the plurality of designed residues over a plurality ofcombinations.

At block 350, the computing device can design a second protein backbonefor the protein for flexibility and low energy structures based on thefirst protein backbone.

At block 360, the computing device can generate an output related to atleast the second protein backbone. In some examples, generating theoutput related to the second protein backbone for the protein caninclude designing one or more molecules based on the second proteinbackbone for the protein.

In other examples, generating the output related to the second proteinbackbone for the protein can include: generating a synthetic gene forthe protein that is based the second protein backbone for the protein;expressing a particular protein in vivo using the synthetic gene; andpurifying the particular protein. In some of these examples, expressingthe particular protein sequence in vivo using the synthetic gene caninclude expressing the particular protein sequence in one or moreEscherichia coli that include the synthetic gene.

In other examples, generating the output related to the second proteinbackbone for the protein can include generating one or more images thatinclude at least part of the second protein backbone for the protein.

In other examples, the computing device can include a protein synthesisdevice; then, generating the output related to at least the secondprotein backbone for the protein can include synthesizing at least thesecond protein backbone for the protein using the protein synthesisdevice.

In one embodiment, the methods are for designing a protein mimetic, asexemplified herein.

Also included are non-naturally occurring proteins prepared by thecomputational methods described herein. The non-naturally occurringproteins can be cytokines, for example, non-naturally occurring IL-2 orIL-4 mimetics.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of embodiments of the present invention only andare presented in the cause of providing what is believed to be the mostuseful and readily understood description of the principles andconceptual aspects of various embodiments of the invention. In thisregard, no attempt is made to show structural details of the inventionin more detail than is necessary for the fundamental understanding ofthe invention, the description taken with the drawings and/or examplesmaking apparent to those skilled in the art how the several forms of theinvention may be embodied in practice.

The above definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3^(rd) Edition or a dictionary known to those of skill inthe art, such as the Oxford Dictionary of Biochemistry and MolecularBiology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

The above description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure. The description of embodiments of thedisclosure is not intended to be exhaustive or to limit the disclosureto the precise form disclosed. While specific embodiments of, andexamples for, the disclosure are described herein for illustrativepurposes, various equivalent modifications are possible within the scopeof the disclosure, as those skilled in the relevant art will recognize.

All of the references cited herein are incorporated by reference.Aspects of the disclosure can be modified, if necessary, to employ thesystems, functions and concepts of the above references and applicationto provide yet further embodiments of the disclosure. These and otherchanges can be made to the disclosure in light of the detaileddescription.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The above detailed description describes various features and functionsof the disclosed systems, devices, and methods with reference to theaccompanying figures. In the figures, similar symbols typically identifysimilar components, unless context dictates otherwise. The illustrativeembodiments described in the detailed description, figures, and claimsare not meant to be limiting. Other embodiments can be utilized, andother changes can be made, without departing from the spirit or scope ofthe subject matter presented herein. It will be readily understood thatthe aspects of the present disclosure, as generally described herein,and illustrated in the figures, can be arranged, substituted, combined,separated, and designed in a wide variety of different configurations,all of which are explicitly contemplated herein.

With respect to any or all of the ladder diagrams, scenarios, and flowcharts in the figures and as discussed herein, each block and/orcommunication may represent a processing of information and/or atransmission of information in accordance with example embodiments.Alternative embodiments are included within the scope of these exampleembodiments. In these alternative embodiments, for example, functionsdescribed as blocks, transmissions, communications, requests, responses,and/or messages may be executed out of order from that shown ordiscussed, including substantially concurrent or in reverse order,depending on the functionality involved. Further, more or fewer blocksand/or functions may be used with any of the ladder diagrams, scenarios,and flow charts discussed herein, and these ladder diagrams, scenarios,and flow charts may be combined with one another, in part or in whole.

A block that represents a processing of information may correspond tocircuitry that can be configured to perform the specific logicalfunctions of a herein-described method or technique. Alternatively oradditionally, a block that represents a processing of information maycorrespond to a module, a segment, or a portion of program code(including related data). The program code may include one or moreinstructions executable by a processor for implementing specific logicalfunctions or actions in the method or technique. The program code and/orrelated data may be stored on any type of computer readable medium suchas a storage device including a disk or hard drive or other storagemedium.

The computer readable medium may also include non-transitory computerreadable media such as computer-readable media that stores data forshort periods of time like register memory, processor cache, and randomaccess memory (RAM). The computer readable media may also includenon-transitory computer readable media that stores program code and/ordata for longer periods of time, such as secondary or persistent longterm storage, like read only memory (ROM), optical or magnetic disks,compact-disc read only memory (CD-ROM), for example. The computerreadable media may also be any other volatile or non-volatile storagesystems. A computer readable medium may be considered a computerreadable storage medium, for example, or a tangible storage device.Moreover, a block that represents one or more information transmissionsmay correspond to information transmissions between software and/orhardware modules in the same physical device. However, other informationtransmissions may be between software modules and/or hardware modules indifferent physical devices.

Numerous modifications and variations of the present disclosure arepossible in light of the above teachings.

Examples

A computational approach for designing de novo cytokine mimetics isdescribed that recapitulate the functional sites of the naturalcytokines, but otherwise are unrelated in topology or amino acidsequence. This strategy was used to design de novo mimetics of IL-2 andinterleukin-15 (IL-15)¹⁵ that bind to the IL-2 receptor βν_(c)heterodimer (IL-2Rβν_(c))^(16,17), but have no binding site for IL-2Rαor IL-15Rα. The designs are hyper-stable, bind to human and mouseIL-2Rβν_(c) with higher affinity than the natural cytokines, and elicitdownstream cell signaling independent of IL-2Rα and IL-15Rα. Crystalstructures of an experimentally optimized mimetic, neoleukin-2/15, arevery close to the design model and provide the first structuralinformation on the murine IL-2Rβν_(c) complex. Neoleukin-2/15 has highlyefficacious therapeutic activity compared to IL-2 in murine models ofmelanoma and colon cancer, with reduced toxicity and no signs ofimmunogenicity. This strategy for building hyper-stable de novo mimeticscan be readily applied to a multitude of natural cytokines and othersignaling proteins, enabling the creation of superior therapeuticcandidates with enhanced clinical profiles.

Because of the potent biological activity of natural protein hormonesand cytokines, there have been extensive efforts to improve theirpotential therapeutic efficacy through protein engineering. Such effortshave sought to simplify manufacturing, extend half-life, and modulatereceptor interactions¹⁸⁻²⁰. However, there are inherent challenges tothe development of a new therapeutic when starting with a naturallyoccurring bioactive protein. First, most natural proteins are onlymarginally stable²¹⁻²⁵, hence amino acid substitutions aimed atincreasing efficacy can decrease expression or cause aggregation, makingmanufacturing and storage difficult. More substantial changes, such asthe deletion or fusion of functional or targeting domains, are oftenunworkable and can dramatically alter pharmacokinetic properties andtissue penetration¹⁹. Second, any immune response against the engineeredvariant may cross-react with the endogenous molecule²⁶⁻³⁵ withpotentially catastrophic consequences. A computational design approachwas developed to generate analogues of natural proteins with improvedtherapeutic properties that circumvent these challenges, focusing efforton engineering de novo cytokine mimetics displaying specific subsets ofthe receptor binding interfaces optimal for treating disease.

Many cytokines interact with multiple different receptorsubunits^(15,16,36-39) and like most naturally occurring proteins,contain non-ideal structural features that compromise stability but areimportant for function. A computational protocol was developed in whichthe structural elements interacting with the desired receptor subunit(s)are fixed in space, and an idealized globular protein structure is builtto support these elements. Previous efforts were extended usingcombinatorial fragment assembly to support short linear epitopes withparametric construction of disembodied helices coupled withknowledge-based loop closure (FIG. 1a-b ). The approach was tested byattempting to de novo design stable idealized proteins with interactionsurfaces mimicking those of human IL-2 (hIL-2) and human IL-15 (hIL-15)for the human IL-2Rβν_(c) (hIL-2Rβν_(c)), but entirely lacking the IL-2receptor alpha (IL-2Rα) interaction surface. Previous efforts atremoving the alpha interaction region in hIL-2, by eithermutation^(9,44,45) (e.g. F42A mutation of Super-2, also known as H9⁹) orPEGylation (e.g. NKTR-214^(9,13)), have resulted in markedly reducedstability, binding and/or potency of the cytokine on the hIL-2Rβν_(c)receptor while failing to completely eliminate the alpha interaction.

Computational Design of IL-2/IL-15 Mimetics that Bind and ActivateIL-2Rβν_(c):

Native hIL-2 comprises four helices connected by long irregular loops.The N-terminal helix (H1) interacts with both the beta and gammasubunits of the IL-2 receptor, the third helix (H3) interacts with thebeta subunit, and the C-terminal helix (H4) with the gamma subunit; thealpha subunit interacting surface is formed by the irregular secondhelix (H2) and two long loops, one connecting H1 to H2 and the otherconnecting H3 and H4. An idealized protein was designed thatrecapitulates the interface formed by H1, H3 and H4 with beta and gammaand to replace H2 with a regular helix that offers better packing. Thehelices H1, H3 and H4 (see FIG. 1a ) were used as a template for thebinding site, while helix H2 was reconstructed (H2′) using a databaseoff highly-represented clustered-fragments (see Methods). Pairs ofhelices were connected with loops extracted from the same database (seeFIG. 1b ), the resulting helical hairpins combined into fully connectedbackbones (see FIG. 1c ), and Rosetta⁴⁶⁻⁴⁸ combinatorial flexiblebackbone sequence design calculations were carried out in the presenceof hIL-2Rβν_(c) (see Methods). The top four computational designs andeight single-disulfide stapled variations (see Table Si) were selectedfor experimental characterization by yeast display (see Methods). Eightdesigns were found to bind fluorescently-tagged beta-gamma chimeric IL-2receptor at low-nanomolar concentrations. The best non-disulfide design(G1 neo2 40) was subjected to site saturation mutagenesis followed byselection and combination of affinity-increasing substitutions for themurine IL-2Rβ

_(c) (mIL-2Rβν_(c), see FIG. 10). Optimized designs (were expressedrecombinantly in E. coli and found to elicit pSTAT5 signaling in vitroon IL-2-responsive murine cells at low-nanomolar or even picomolarconcentrations (see Table E1), but had relatively low thermal stability(Tm ˜<45° C., see FIGS. 14 and 15). To improve stability, thecomputational design protocol was repeated starting from the backbone ofthe highest affinity first round design (G1_neo2_40_1F, topology:H1->H4->H2′->H3), coupling the loop building process with parametricvariation in helix length (+/−8 amino acids, see FIG. 1a bottom panel).This second approach improved the quality of the models by enabling theexploration of substantially more combinations of loops connecting eachpair of helices. The fourteen best designs of the second generation,along with twenty-seven Rosetta sequence redesigns of G1_neo2_40_1F (seeTable S3), were experimentally characterized and all but one were foundto bind IL-2 receptor at low-nanomolar concentrations (FIG. 1d , TableE1, and FIG. 16). The three highest affinity and stability designs (onesequence redesign and two new mimetics) were subjected to sitesaturation mutagenesis for mIL-2Rβν_(c) binding (FIGS. 11-13), followedby selection and combination of affinity-increasing substitutions forboth human and mouse IL-2Rβν_(c). The matured designs (see Table S4)showed enhanced binding while retaining hyper-stability (see Table E1).The top design, neoleukin-2/15 (also referred to herein as Neo-2/15), isa 100 residue protein with a new topology and sequence quite differentfrom human or murine IL-2 (29% sequence identity to hIL-2 over 89residues, and 16% sequence identity to mIL-2 over 76 aligned residues,in structural topology-agnostic based alignment, see Table E1).

Functional Characterization of Neoleukin-2/15:

Neoleukin-2/15 binds with high affinity to human and mouse IL-2Rβν_(c)(Kd ˜38 nM and ˜19 nM, respectively), but does not interact with IL-2Rα(FIG. 2a ). The affinities of Neoleukin-2/15 for the human and mouseIL-2 receptors (IL-2Rβ and IL-2Rβν_(c)) are significantly higher thanthose of the corresponding native IL-2 cytokines. In contrast withnative IL-2, Neoleukin-2/15 elicits IL-2Rα-independent signaling in bothhuman and murine IL-2-responsive cells (FIG. 2b , top), and in murineprimary T cells (FIG. 2b , bottom). Neoleukin-2/15 activates IL-2Rα−cells more potently than native human or murine IL-2 in accordance withits higher binding affinity. In primary cells, neoleukin-2/15 is moreactive on IL-2Rα− cells and less active on IL-2Rα+ compared to Super-2,presumably due to its complete lack of IL-2Rα binding. Neoleukin-2/15 ishyper-stable (see FIG. 17) and does not lose binding affinity forhIL-2Rβν_(c) following incubation at 80° C. for 2 hours, while hIL-2 andSuper-2 are completely inactivated after 10 minutes (half-inactivationtime=˜4.2 min and ˜2.6 min, respectively, FIG. 2c ). Similarly, in exvivo primary cell cultures, neoleukin-2/15 drove T cell survivaleffectively after being boiled for 60 minutes at 95° C., while theseconditions inactivated both IL-2 and Super-2 (FIG. 2c , bottom). Thermaldenaturation studies were carried out on many other of the designedmimetics, demonstrating their thermal stability as well (see FIG.14-16). This unprecedented stability for a cytokine-like molecule,beyond eliminating the requirement for cold chain storage, suggests arobustness to mutations (see FIGS. 13 and 18-19), genetic fusions andchemical modification greatly exceeding that of native IL-2, which couldcontribute to the development of improved or new therapeutic properties(see FIG. 7).

Structure of Monomeric Neoleukin-2/15 and Ternary Complex withmIL-2Rβν_(c):

The X-ray crystal structure of neoleukin-2/15 was determined and foundit to be very close to the computational design model(r.m.s.d._(Cα)=1.1-1.3 Å for the 6 copies in the asymmetric unit, FIG.3a ). The crystal structure of neoleukin-2/15 in a ternary complex withmurine IL-2Rβν_(c) (FIG. 3b , Table E2) was solved; this may be thefirst example in which a de novo designed protein enabled the structuraldetermination of a previously unsolved natural receptor complex. Theneoleukin-2/15 design model and crystal structure align with the mouseternary complex structure with r.m.s.d._(Cα) of 1.27 and 1.29 Å,respectively (FIG. 3c ). The order of helices in Neoleukin-2/15 (in IL-2numbering) is H1->H3->H2′->H4 (see FIGS. 1a and 3 a,d). The H1-H3 loopis disordered in the ternary complex, but helix H3 is in close agreementwith the predicted structure; there is also an outward movement of helixH4 and the H2′-H4 loop compared to the monomeric structure (FIG. 3c ).Neoleukin-2/15 interacts with mIL-2Rβ via helices H1 and H3, and withν_(c) via the H1 and H4 helices (FIG. 3c ), and these regions alignclosely with both the computational design model (FIG. 3a ) and themonomeric crystal structure (FIG. 3c ). Structural alignment to thepreviously reported crystal structure of the hIL-2 receptor complex⁴⁹reveals a close agreement between the helical backbones ofNeoleukin-2/15 and hIL-2 in the binding site, despite the differenttopology of the two proteins (FIG. 3d-e ). Some side chain interactionsbetween neoleukin-2/15 and mIL-2Rβν_(c) are present in thehIL-2-hIL-2Rβνcomplex, while others such as L19Y, arose during thecomputational design process.

Therapeutic Applications of Neoleukin-2/15:

The clinical use of IL-2 has been mainly limited by toxicity⁵⁰⁻⁵².Although the interactions responsible for IL-2 toxicity in humans areincompletely understood, in murine models toxicity is T cell independentand ameliorated in animals deficient in the IL-2Rα chain (CD25+). Thus,many efforts have been directed to reengineer IL-2 to weakeninteractions with IL-2Rα, but mutations in the CD25 binding site can behighly destabilizing⁶. The inherent low stability of IL-2 and itstightly evolved dependence on CD25 have been barriers to the translationof reengineered IL-2 compounds. Other efforts have focused onIL-15^(53,54), since it elicits similar signaling to IL-2 by dimerizingthe IL-2Rβν_(c) but has no affinity for CD25. However, IL-15 isdependent on trans presentation by the IL-15α (CD215) receptor that isdisplayed primarily on antigen-presenting cells and natural killercells. The low stability of native IL-15 and its dependence on transpresentation have also been substantial barriers to reengineeringefforts⁵³⁻⁵⁵.

Dose escalation studies on naive mice show that mIL-2 preferentiallyexpands regulatory T cells, consistent with preferential binding toCD25+ cells^(41,56,57) while neoleukin-2/15 primarily drives expansionof CD8⁺ T cells (FIG. 4a ) and does not induce or minimally inducesexpansion of regulatory T cells only at the highest dose tested.Similarly, in a murine model of airway inflammation, which normallyinduces a small percentage of tissue resident CD8+ T cells,neoleukin-2/15 produces an increase in Thy1.2⁻ CD44⁺ CD8⁺ T cellswithout increasing CD4⁺ Foxp3⁺ antigen-specific Tregs in the lymphoidorgans (FIG. 4b ).

De novo protein design allows the circumvention of the structurallimitations of native cytokines, but there is a possibility of elicitinganti-drug antibodies. To test whether neoleukin-2/15 elicits ananti-drug response, tumor-bearing mice were treated daily withneoleukin-2/15 over a period of 2 weeks, and no evidence of anti-drugantibodies was observed in any of the treated animals (FIG. 4c , leftpanel; a similar lack of immune response was observed for other de novodesign therapeutic candidates⁴¹). Polyclonal antibodies againstneoleukin-2/15 were produced by vaccinating mice with an inactiveneoleukin-2/15 mutant (K.O. neoleukin) in complete Freund's adjuvant.These polyclonal anti-neoleukin-2/15 antibodies did not cross react withhuman or mouse IL-2 (FIG. 4c ). The absence of binding to native IL-2suggests that even if there is an immune response to neoleukin-2/15,this response is unlikely to cross-react with endogenous IL-2.Furthermore, since the sequence identity between neoleukin-2/15 andhIL-2 is low (<30%, see Table E1), an autoimmune response against hostIL-2 is much more likely with previous engineered hIL-2 variants (e.g.Super-2, see Table E1) which differ from endogenous IL-2 by only a fewmutations.

The therapeutic efficacy of neoleukin-2/15 was tested in the poorlyimmunogenic B16F10 melanoma and the more immunogenic CT26 colon cancermouse models. Single agent treatment with neoleukin-2/15 led todose-dependent delays in tumour growth in both cancer models. In CT26colon cancer, single agent treatment showed improved efficacy to thatobserved for recombinant mIL-2 (FIG. 4d and FIG. 5). In B16F10 melanoma,co-treatment with the anti-melanoma antibody TA99 (anti-TRP1) led tosignificant tumour growth delays, while TA99 treatment alone had littleeffect (FIG. 4e and FIG. 6). In long term survival experiments (8weeks), neoleukin-2/15 in combination with TA99 showed substantiallyreduced toxicity and an overall superior therapeutic effect compared tomIL-2 (FIG. 4e ). Mice treated with the combination mIL-2 and TA99steadily lost weight and their overall health declined to the point ofrequiring euthanasia, whereas little decline was observed with thecombination of neoleukin-2/15 and TA99 (FIG. 4e ). Consistent with atherapeutic benefit, neoleukin-2/15 treatment led to a significantincrease in intratumoral CD8:T_(reg) ratios (see FIG. 4f and FIG. 5),which has been previously correlated with effective antitumor immuneresponses⁵⁸. The increases of CD8:T_(reg) ratios by neoleukin-2/15 aredose and antigen dependent (FIG. 40; optimum therapeutic effects wereobtained at higher doses and in combination with other immunotherapies(see FIG. 6). Altogether, these data show that neoleukin-2/15 exhibitsthe predicted homeostatic benefit derived from its IL-2 likeimmunopotentiator activity, but without the adverse effects associatedwith CD25⁺ preferential binding. These enhanced properties andlow-toxicity may allow the routine use of neoleukin-2/15 for otherimmunotherapies where recombinant IL-2 is not broadly used. As anexample of such a use, the potential application of neoleukin-2/15 toenhance CAR-T cell therapy (see FIG. 8) was investigated. NSG miceinoculated with 0.5×10⁶ RAJI tumor cells were left untreated, weretreated with 0.8×10⁶ anti-CD19 CAR-T cells (infused 7 days afterinoculation of tumor cells), or were similarly treated with anti-CD19CAR-T cells plus 20 μg/day of either human IL-2 or neoleukin-2/15 ondays 8-14 after tumor inoculation. As expected, Neoleukin-2/15significantly enhanced the anti-tumor effect of CAR-T cell therapy inthis model, slowing growth of the tumor and extending the survival ofthe mouse (data not shown).

De novo design of protein mimetics has the potential to transform thefield of protein-based therapeutics, enabling the development ofbiosuperior molecules with enhanced therapeutic properties and reducedside-effects, not only for cytokines, but for virtually any biologicallyactive molecule with known or accurately predictable structure. Becauseof the incremental nature of current traditional engineering approaches(e.g. 1-3 amino acid substitutions, chemical modification at a singlesite), most of the shortcomings of the parent molecule are inevitablypassed on to the resulting engineered variants, often in an exacerbatedform. By building mimetics de novo, these shortcomings can be completelyavoided: unlike recombinant IL-2 and engineered variants of hIL-2,neoleukin-2/15 can be solubly expressed in E. coli (see FIG. 17),retains activity at high temperature, does not interact with IL-2Rα andis robust to substantial sequence changes that allow the engineering ofnew functions (FIG. 7). Likely because of the small size and highstability of de novo designed proteins, immunogenicity appears to below, and in contrast to incremental variants of hIL-2, any antibodyresponse to the mimetic is unlikely to cross react with the naturalparent cytokine. Because of their high stability and robustness, andtheir tailored interaction surfaces, designed mimetics are likely to beparticularly powerful in next generation therapeutics which combinedifferent protein functionalities, for example targeted versions ofneoleukin-2/15.

Robust Modularity of Neoleukin-2/15. Disulfide-Stapling andReengineering into an IL-4 Mimetic:

Neoleukin-2/15 is highly modular, allowing to easily tune itsproperties, such as increasing its stability or modify its bindingpreference. This modularity and robustness was taken advantage of byintroducing, by computational design, stability enhancingsingle-disulfide staples that preserve the function of neoleukin-2/15⁵⁹.For this, two orthogonal strategies were used. First, a disulfide bridgewas introduced by searching pairs of positions with favorablegeometrical arrangements followed by flexible backbone minimization. Thefinal design introduced a single disulfide between residues 38 and 75,which stabilizes helices H3 and H2. In the second approach, the N- andC-terminus of neoleukin-2/15 was remodeled to allow the introduction ofa single-disulfide staple that encompasses the entire protein (addedsequences CNSN (SEQ ID NO:260) and NFQC (SEQ ID NO:261), for N- andC-termini, respectively after removing terminal P and S residues, seeFIG. 18). Both disulfide stapling strategies increased the stability ofneoleukin-2/15 (Tm>95° C.), while retaining its sequence and functionmostly unaffected (see FIG. 18). The modularity properties ofneoleukin-2/15 were used to modify its binding preference. All cytokinesin the interleukin-2 family interact with the ν_(c) and share a commonarchitecture. Therefore, it was hypothesized that neoleukin-2/15 couldbe transformed into another cytokine mimetic of the IL-2 family bychanging only amino acids in the half of the binding-site that interactswith IL-2Rβ (helices H1 and H3). As proof of a concept, humaninterleukin-4 (hIL-4) was chosen as target, since it shares extensivestructural homology with IL-2 and has potential applications inregenerative medicine^(60,61). Neo-2/15 was modified to bind to thehuman IL-4 receptor (comprising IL-4Rα and ν_(c)) and not to the humanIL-2 receptor (comprising IL-2Rβ and ν_(c)) by aligning the Neo-2/15model into the structure of human IL-4 bound to its IL-4 receptor, andmutating 14 residues in Neo-2/15 to match the amino-acids of IL-4 atthose structural positions that mediate interactions between IL-4 andIL4r (FIG. 7). Binding was further optimized by directed evolution usingrandom mutagenesis and screening for high binding affinity variants,which introduced two additional amino acid substitutions and modifiedone of the fourteen original residues grafted from the IL-4 protein,thereby creating a new protein Neoleukin-4 with a total of sixteenmutations from Neoleukin-2/15. The resulting optimized design,neoleukin-4 (see Table S5), was recombinantly expressed and purifiedfrom E. coli and tested for binding. Neoleukin-4 binds with highaffinity to IL-4Rα receptor, binds cooperatively to IL-4Rαν_(c) (seeFIG. 7), and does not bind with any affinity to the IL-2 receptor (datanot shown) Neoleukin-4 retains the superior thermostable properties ofneoleukin-2/15 (see FIG. 20b,c ), and binds to the IL-13 receptor asexpected given the natural cross-reactivity of IL-4 to IL-13 receptor(data not shown). Altogether, this shows that neoleukin-2/15 is robustenough to act as a modular scaffold where significant rational sequencechanges can be introduced to modify its function or physical propertiesin a highly predictable way

Methods

Computational Design of De Novo Cytokine Mimetics:

The design of de novo cytokine mimetics began by defining a thestructure of hIL-2 in the quaternary complex with the IL-2Rβν_(c)receptor as template for the design. After inspection, the residuescomposing the binding-site were defined as hotspots using Rosetta'smetadata (PDBInfoLabels). The structure was feed into the new mimeticdesign protocol that is programmed in PyRosetta, and which canautomatically detect the core-secondary structure elements that composethe target-template and produce the resulting de novo mimetic backboneswith full RosettaScripts compatible information for design. Briefly, themimetic building algorithm works as follows. For the first generation ofdesigns, each of the core-elements was idealized by reconstruction usingloops from a clustered database of highly-ideal fragments (fragment-size4 amino acids). After idealization, the mimetic building protocol aimsto reconnect the idealized elements by pairs in all possiblecombinations. To do this it uses combinatorial fragment assembly ofsequence-agnostic fragments from the database, followed bycartesian-constrained backbone minimization for potential solutions(i.e. where the N- and C-ends of the built fragment are close enough tolink the two secondary structures). After minimization, the solutionsare verified to contain highly ideal fragments (i.e. that everyoverlapping fragment that composes the two connected elements is alsocontained within the database) and no backbone clashes with the target(context) receptor. Passing backbone solutions were then profiled usingthe same database of fragments in order to determine the most probableamino acids at each position (this information was encoded in metadataon the design). Next, solutions for pairs of connected secondarystructures were combinatorially recombined to produce fully connectedbackbones by using graph theory connected components. Since the numberof solutions grows exponentially with each pair of elements, at eachfragment combination step we ranked the designs to favor those withshorter interconnections between pairs of core elements, and kept onlythe top solutions to proceed to the next step. Fully connected solutionswere then profiled by layer (interface, core, non-core-surface,surface), in order to restrict the identities of the possible aminoacids to be layer-compatible. Finally, all the information on hotspots,compatible built-fragment amino acids and layers were combined (hotspothas precedence to amino acid probability, and amino acid probabilitytook precedence to layer). These fully profiled backbones were thenpassed to RosettaScripts for flexible backbone design and filtering (seerosetta-script in Appendix A). For the second generation of designs, twoapproaches were followed. In the first approach, sequence redesigns ofthe best first generation optimized design were executed (G1_neo2_40_1F,see Appendix B). In the second approach new mimetics were engineeredusing G1_neo2_40_1F as the target template. The mimetic design protocolin this second generation was similar to the one described for the firstgeneration, but with two key differences. Firstly, the core-fragmentswere no longer built from fragments, but instead by discoveringparametric equations of repetitive phi and psi angles (omega fixed to180°) that result in repetitive secondary structures that recapitulatedeach of the target helices as close as possible, a “pitch” on the phiand psi angles was allowed every X-amino acids in order to allow thehelices the possibility to have curvature (final parameters: H1:, H2:,H3, H4), the sue of these parametric equations allowed to change thesize of each of the core-elements in the target structure at will(either increase or decrease the size), which was coupled (max/min8.a.a.) with the loop building process, and reductions in the size ofthe core elements were not allowed to remove hotspots from the bindingsite. The second difference in the second generation designs, is thatinstead of reconnecting the secondary structure core-elements we used afragment-size of 7 amino acids, and no combinatorial assembly of morethan one fragment was allowed (i.e. a single fragment has to be able toclose a pair of secondary structures). The rest of the design algorithmwas in essence similar to the one followed in the generation one (seeAppendix C). The Rosetta energy functions used were “talaris2013” and“talaris2014”, for the first and second generation of designs,respectively.

The databases of highly ideal fragments used for the design of thebackbones for the de novo mimetics were constructed with the new Rosettaapplication “kcenters_clustering_of_fragments” using an extensivedatabase of non-redundant publicly available protein structures from theRCSB protein data bank, which was comprised of 16767 PDBs for the 4-merdatabase used for the first generation designs, and 7062 PDBs for the7-mer database used for the second generation designs.

Yeast Display:

Yeast were transformed with genes encoding the proteins to be displayedtogether with linearized pETcon3 vector. The vector was linearized by100 fold overdigestion by NdeI and XhoI (New England Biolabs) and thenpurified by gel extraction (Qiagen). The genes included 50 bases ofoverlap with the vector on both the 5′ and 3′ ends such that homologousrecombination would place the genes in frame between the AGA2 gene andthe myc tag on the vector. Yeast were grown in C-Trp-Ura media prior toinduction in SGCAA media as previously described. 12-18 hours afterinduction, cells were washed in chilled display buffer (50 mM NaPO₄ pH8, 20 mM NaCl, 0.5% BSA) and incubated with varying concentrations ofbiotinylated receptor (either human or murine IL-2Rα, IL-2Rβ, IL-2Rν, orhuman IL-4Rα) while being agitated at 4° C. After approximately 30minutes, cells were washed again in chilled buffer, and then incubatedon ice for 5 minutes with FITC-conjugated anti-c-Myc antibody (1 uL per3×10⁶ cells) and streptavidin-phycoerythrin (1 uL per 100 uL volume ofyeast). Yeast were then washed and counted by flow cytometry (Accuri C6)or sorted by FACS (Sony SH800). For experiments in which the initialreceptor incubation was conducted with a combination of biotinylatedIL-2Rν and non-biotinylated IL-4Rα, the non-biotinylated receptor wasprovided in molar excess.

Mutagenesis and Affinity Maturation:

For error-prone PCR based mutagenesis, the design to be mutated wascloned into pETcon3 vector and amplified using the MutaGene IImutagenesis kit (Invitrogen) per manufacturer's instructions to yield amutation frequency of approximately 1% per nucleotide. 1 μg of thismutated gene was electroporated into EBY100 yeast together with 1 μg oflinearized pETcon3 vector, with a transformation efficiency on the orderof 10⁸. The yeast were induced and sorted multiple times in successionwith progressively decreasing concentrations of receptor untilconvergence of the population. The yeast were regrown in C-Trp-Ura mediabetween each sort.

Site-saturation mutagenesis (SSM) libraries were constructed fromsynthetic DNA from Genscript. For each amino acid on each designtemplate, forward primers and reverse primers were designed such thatPCR amplification would result in a 5′ PCR product with a degenerate NNKcodon and a 3′ PCR product, respectively. Amplification of “left” and“right” products by COF and COR primers yielded a series of templateproducts each consisting of a degenerate NNK codon at a differentresidue position. For each design, these products were pooled to yieldthe SSM library. SSM libraries were transformed by electroporation intoconditioned Saccharomyces cerevisiae strain EBY100 cells, along withlinearized pETCON3 vector, using the protocol previously described byBenatuil et al.

Combinatorial libraries were constructed from synthetic DNA fromGenscript containing ambiguous nucleotides and similarly transformedinto linearized pETCON3 vector.

Protein Expression:

Genes encoding the designed protein sequences were synthesized andcloned into pET-28b(+) E. coli plasmid expression vectors (GenScript,N-terminal 6×His tag and thrombin cleavage site). Plasmids were thentransformed into chemically competent E. coli Lemo21 cells (NEB).Protein expression was performed using Terrific Broth and M salts,cultures were grown at 37° C. until OD⁶⁰⁰ reached approximately 0.8,then expression was induced with 1 mM of isopropylβ-D-thiogalactopyranoside (IPTG), and temperature was lowered to 18° C.After expression for approximately 18 hours, cells were harvested andlysed with a Microfluidics M110P microfluidizer at 18,000 psi, then thesoluble fraction was clarified by centrifugation at 24,000 g for 20minutes. The soluble fraction was purified by Immobilized Metal AffinityChromatograpy (Qiagen) followed by FPLC size-exclusion chromatography(Superdex 75 10/300 GL, GE Healthcare). The purified neoleukin-2/15 wascharacterized by Mass Spectrum (MS) verification of the molecular weightof the species in solution (Thermo Scientific), SizeExclusion-MultiAngle Laser Light Scattering (SEC-MALLS) in order toverify monomeric state and molecular weight (Agilent, Wyatt), SDS-PAGE,and endotoxin levels (Charles River).

Human and mouse IL-2 complex components including hIL-2 (a.a. 1-133),hIL-2Rα (a.a. 1-217), hIL-2Rβ (a.a. 1-214) hIL-2Rγ (a.a. 1-232), mIL-2(a.a. 1-149), mIL-2Rα ectodomain (a.a. 1-213), mIL-2Rβ ectodomain (a.a.1-215), and my, ectodomain (a.a. 1-233) were secreted and purified usinga baculovirus expression system, as previously described^(17,49). Allproteins were purified to >98% homogeneity with a Superdex 200 sizingcolumn (GE Healthcare) equilibrated in HBS. Purity was verified bySDS-PAGE analysis. For expression of biotinylated human IL-2 and mouseIL-2 receptor subunits, proteins containing a C-terminal biotin acceptorpeptide (BAP)-LNDIFEAQKIEWHE (SEQ ID NO:262) were expressed and purifiedas described via Ni-NTA affinity chromatography and then biotinylatedwith the soluble BirA ligase enzyme in 0.5 mM Bicine pH 8.3, 100 mM ATP,100 mM magnesium acetate, and 500 mM biotin (Sigma). Excess biotin wasremoved by size exclusion chromatography on a Superdex 200 columnequilibrated in HBS.

Neoleukin-2 Crystal and Co-Crystal Structures:

C-terminally 6×His-tagged endoglycosidase H (endoH) and murine IL-2Rβand IL-2Rγ were expressed separately in Hi-five cells using abaculovirus system as previously described. IL-2Rγ was grown in thepresence of 5 μM kifunensin. After approximately 72 hours, the secretedproteins were purified from the media by passing over a Ni-NTA agarosecolumn and eluted with 200 mM imidazole in HBS buffer (150 mM NaCl, 10mM HEPES pH 7.3). EndoH was exchanged into HBS buffer by diafiltration.mIL-2Rγ was deglycosylated by overnight incubation with 1:75 (w/w)endoH. mIL-2Rβ and mIL-2Rγ were further purified and buffer exchanged byFPLC using an S200 column (GE Life Sciences).

Monomeric neoleukin-2/15 was concentrated to 12 mg/ml and crystallizedby vapor diffusion from 2.4 M sodium malonate pH 7.0, and crystals wereharvested and flash frozen without further cryoprotection. Crystalsdiffracted to 2.0 Å resolution at Stanford Synchrotron RadiationLaboratory beamline 12-2 and were indexed and integrated using XDS(Kabsch, 2010). The space group was assigned with Pointless (Evans,2006), and scaling was performed with Aimless (Evans and Murshudov,2013) from the CCP4 suite (Winn et al., 2013). Our predicted model wasused as a search ensemble to solve the structure by molecularreplacement in Phaser (McCoy et al., 2007), with six protomers locatedin the asymmetric unit. After initial rebuilding with Autobuild(Terwilliger et al., 2008), iterative cycles of manual rebuilding andrefinement were performed using Coot (Emsley et al., 2010) and Phenix(Adams et al., 2010).

To crystallize the ternary neoleukin:mIL-2Rβ:mIL-2Rγ complex, the threeproteins were combined in equimolar ratios, digested overnight with1:100 (w/w) carboxypeptidases A and B to remove purification tags, andpurified by FPLC using an 5200 column; fractions containing all threeproteins were pooled and concentrated to 20 mg/ml. Initial needlelikemicrocrystals were formed by vapor diffusion from 0.1 M imidazole pH8.0, 1 M sodium citrate and used to prepare a microseed stock forsubsequent use in microseed matrix screening (MMS, (D'Arcy et al.,2014)). After a single iteration of MMS, crystals grown in the sameprecipitant were cryoprotected with 30% ethylene glycol, harvested anddiffracted anisotropically to 3.4 Å×3.8 Å×4.1 Å resolution at AdvancedPhoton Source beamline 231D-B. The structure was solved by molecularreplacement in Phaser using the human IL-2Rβ and IL-2Rγ structures (pdbID 2B51) as search ensembles. This produced an electron density map intowhich two poly-alanine alpha helices could be manually built. Followingrigid body refinement in Phenix, electron density for the two unmodeledalpha helices, along with the BC loop and some aromatic side chains,became visible, allowing docking of the monomeric neoleukin. Two furtheriterations of MMS and use of an additive screen (Hampton Research)produced crystals grown by vapor diffusion using 150 nl of protein, 125nl of well solution containing 0.1 M Tris pH 7.5, 5% dextran sulfate,2.1 M ammonium sulfate and 25 nl of microseed stock containing 1.3 Mammonium sulfate, 50 mM Tris pH 7.5, 50 mM imidazole pH 8.0, 300 mMsodium citrate. Crystals cryoprotected with 3 M sodium malonate wereflash frozen and diffracted anisotropically to 2.5 Å×3.7 Å×3.8 Å atAdvanced Light Source beamline 5.0.1. After processing the data withXDS, an elliptical resolution limit was applied using the STARANISOserver (Bruhn et al., 2017). Rapid convergence of the model was obtainedby refinement against these reflections using TLS and target restraintsto the higher resolution human receptor (PDB id 2B51) and neoleukin-2/15structures in Buster (Smart et al., 2012; Bricogne et al., 2016), withmanual rebuilding in Coot, followed by a final round of refinement inPhenix with no target restraints. Structure figures were prepared withPyMol (Schrodinger, LLC. 2010. The PyMOL Molecular Graphics System,Version 2.1.0). Software used in this project was installed andconfigured by SBGrid (Morin et al., 2013).

Cell Lines:

Unmodified YT-1⁶⁴ and IL-2Rα⁺ YT-1 human natural killer cells⁶⁵ werecultured in RPMI complete medium (RPMI 1640 medium supplemented with 10%fetal bovine serum, 2 mM L-glutamine, minimum non-essential amino acids,sodium pyruvate, 25 mM HEPES, and penicillin-streptomycin [Gibco]).CTLL-2 cells purchased from ATCC were cultured in RPMI complete with 10%T-STIM culture supplement with ConA (Corning). All cells were maintainedat 37° C. in a humidified atmosphere with 5% CO₂. The subpopulation ofYT-1 cells expressing IL-2Rα was purified via magnetic selection asdescribed previously¹⁷. Enrichment and persistence of IL-2Rα expressionwas monitored by analysis of PE-conjugated anti-human IL-2Rα (Biolegend)antibody binding on an Accuri C6 flow cytometer (BD Biosciences).

Circular Dichroism (CD):

Far-ultraviolet CD measurements were carried out with an AVIVspectrometer model 420 in PBS buffer (pH 7.4) in a 1 mm path-lengthcuvette with protein concentration of ˜0.20 mg/ml (unless otherwisementioned in the text). Temperature melts where from 25 to 95° C. andmonitored absorption signal at 222 nm (steps of 2° C./min, 30 s ofequilibration by step). Wavelength scans (195-260 nm) were collected at25° C. and 95° C., and again at 25° C. after fast refolding (˜5 min).

Binding Studies:

Surface plasmon resonance (SPR): For IL-2 receptor affinity titrationstudies, biotinylated human or mouse IL-2Rα, IL-2Rβ, and IL-2Rγreceptors were immobilized to streptavidin-coated chips for analysis ona Biacore T100 instrument (GE Healthcare). An irrelevant biotinylatedprotein was immobilized in the reference channel to subtractnon-specific binding. Less than 100 response units (RU) of each ligandwas immobilized to minimize mass transfer effects. Three-fold serialdilutions of hIL-2, mIL-2, Super-2, or engineered IL-2 mimetics wereflowed over the immobilized ligands for 60 s and dissociation wasmeasured for 240 s. For IL-2Rγ_(c) binding studies, saturatingconcentrations of hIL-2Rβ (3 uM) or mIL-2Rβ

(5 uM) were added to the indicated concentrations of hIL-2 or mIL-2,respectively. Surface regeneration for all interactions was conductedusing 15 s exposure to 1 M MgCl2 in 10 mM sodium acetate pH 5.5. SPRexperiments were carried out in HBS-P+ buffer (GE Healthcare)supplemented with 0.2% bovine serum albumin (BSA) at 25° C. and allbinding studies were performed at a flow rate of 50 L/min to preventanalyte rebinding. Data was visualized and processed using the BiacoreT100 evaluation software version 2.0 (GE Healthcare). Equilibriumtitration curve fitting and equilibrium binding dissociation (KD) valuedetermination was implemented using GraphPad Prism assuming all bindinginteractions to be first order. Biolayer interferometry: binding datawere collected in a Octet RED96 (ForteBio, Menlo Park, Calif.) andprocessed using the instrument's integrated software using a 1:1 bindingmodel. Biotinylated target receptors, either human or murine IL-2Rα,IL-2Rβ, IL-2Rν, or human IL-4Rα, were functionalized to streptavidincoated biosensors (SA ForteBio) at 1 μg/ml in binding buffer (10 mMHEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5%non-fat dry milk) for 300 seconds. Analyte proteins were diluted fromconcentrated stocks into binding buffer. After baseline measurement inbinding buffer alone, the binding kinetics were monitored by dipping thebiosensors in wells containing 100 nM of the designed protein(association) and then dipping the sensors back into baseline wells(dissociation). For binding experiments in which either IL-2Rβ or IL-4Rαwere supplemented in solution while IL-2Rν was bound to the sensor, thesupplemental proteins were provided in 2.5 fold molar excess

STAT5 Phosphorylation Studies:

In vitro studies: Approximately 2×10⁵ YT-1, IL-2Rα⁺ YT-1, or CTLL-2cells were plated in each well of a 96-well plate and re-suspended inRPMI complete medium containing serial dilutions of hIL-2, mIL-2,Super-2, or engineered IL-2 mimetics. Cells were stimulated for 15 minat 37° C. and immediately fixed by addition of formaldehyde to 1.5% and10 min incubation at room temperature. Permeabilization of cells wasachieved by resuspension in ice-cold 100% methanol for 30 min at 4° C.Fixed and permeabilized cells were washed twice with FACS buffer(phosphate-buffered saline [PBS] pH 7.2 containing 0.1% bovine serumalbumin) and incubated with Alexa Fluor® 647-conjugated anti-STAT5 pY694(BD Biosciences) diluted in FACS buffer for 2 hours at room temperature.Cells were then washed twice in FACS buffer and MFI was determined on aCytoFLEX flow cytometer (Beckman-Coulter). Dose-response curves werefitted to a logistic model and half-maximal effective concentration(EC₅₀ values) were calculated using GraphPad Prism data analysissoftware after subtraction of the mean fluorescence intensity (MFI) ofunstimulated cells and normalization to the maximum signal intensity.Experiments were conducted in triplicate and performed three times withsimilar results. Ex vivo studies: Spleens and lymph nodes were harvestedfrom wild-type C57BL/6J or B6;129S4-Il2ra^(tm1Dw) (CD25KO) micepurchased from The Jackson Laboratory and made into a single cellsuspension in sort buffer (2% Fetal Calf Serum in pH 7.2phosphate-buffered saline). CD4+ T cells were enriched through negativeselection by staining the cell suspension with biotin-conjugatedanti-B220, CD8, NK1.1, CD11b, CD11c, Ter119, and CD19 antibodies at1:100 for 30 min on ice. Following a wash with sort buffer, anti-biotinMicroBeads (Miltenyi Biotec) were added to the cell suspension at 20 μLper 10⁷total cells and incubated on ice for 20 minutes. Cells werewashed, resuspended and negative selection was then performed usingEasySep Magnets (STEMCELL Technologies). Approximately 1×10⁵ enrichedcells were added to each well of a 96-well plate in RPMI complete mediumwith 5% FCS with 10-fold serial dilutions of mIL-2, Super-2, orNeoleukin-2/15. Cells were stimulated for 20 minutes at 37° C. in 5%CO₂, fixed with 4% PFA and incubated for 30 minutes at 4° C. Followingfixation, cells were harvested and washed twice with sort buffer andagain fixed in 500 μL 90% ice-cold methanol in dH₂O for 30 minutes onice for permeabilization. Cells were washed twice with Perm/Wash Buffer(BD Biosciences) and stained with anti-CD4-PerCP in Perm/Wash buffer(1:300). anti-CD44-Alexa Fluor 700 (1:200), anti-CD25-PE-Cy7 (1:200),and 5 μL per sample of anti-pSTAT5-PE pY694 for 45 min at roomtemperature in the dark. Cells were washed with Perm/Wash andre-suspended in sort buffer for analysis on a BD LSR II flow cytometer(BD Biosciences).

In Vivo Murine Airway Inflammation Experiments:

C57BL/6J were purchased from The Jackson Laboratory. Mice wereinoculated intranasally with 20 μL of whole house dust mite antigen(Greer) resuspended in PBS to a total of 23 μg Derp1 per mouse. FromDays 1-7, mice were given a daily intraperitoneal injection of 20 μgmIL-2 in sterile PBS (pH 7.2), a molar equivalent of Neoleukin-2/15 insterile PBS, or no injection. On Day 8, circulating T cells wereintravascularly labeled and tetramer positive cells were enriched fromlymph nodes and spleen or lung as previously described (Hondowicz,Immunity, 2016). Both the column flow-through and bound fractions weresaved for flow cytometry analysis. Cells were surface stained withantibodies and analyzed on a BD LSR II flow cytometer (BD Biosciences)Animal models: C57BL/6 mice were purchased from The Jackson Laboratoryor bred in house and BALB/c mice were purchased from Charles River.Animals were maintained according to protocols approved by Dana-FarberCancer Institute (DFCI) Institutional Animal Care and Use Committee,Direção Geral de Veterinária and iMM Lisboa ethical committee.

Colorectal Carcinoma In Vivo Mice Experiments:

CT26 cells were sourced from Jocelyne Demengeot's research group at IGC(Instituto Gulbenkian de Ciência), Portugal. On day 0, 5×10{circumflexover ( )}5 cells were injected subcutaneously (s.c.) into the flanks ofBALB/c mice with 50 μL of a 1:1 mixture of Dulbecco's modified Eaglemedium (Gibco) with Matrigel (Corning). Starting on day 6, when tumourvolume reached around 100 mm3, neoleukin-2/15 and mIL-2 (Peprotech) wereadministered daily by intraperitoneal (i.p.) injection in 50 μL of PBS(Gibco). Treatment with anti-PD-1 antibody (Bio X Cell) was performedtwice a week by i.p. injection of 200 μg per mouse in PBS. Mice weresacrificed when tumour volume reached 1,300 mm3.

Melanoma In Vivo Experiments:

B16F10 cells were purchased from ATCC. On day 0, 5×10⁵ cells wereinoculated by s.c. injection in 500 μL of Hank's Balanced Salt Solution(Gibco). Starting on day 1, neoleukin-2/15 and mIL-2 (Peprotech) wereadministered daily by intraperitoneal (i.p.) injection in 200 μL ofLPS-free PBS (Teknova). Treatment with TA99 (a gift from Noor Momin andDane Wittrup, Massachusetts Institute of Technology) at 150 μg/mouse wasadded several days later as indicated. Mice were sacrificed when tumorvolume reached 2,000 mm3.

How Cytometry:

Excised tumors were minced, enzymatically digested (Miltenyi Biotec),and passed through a 40-nm filter. Cells from spleens and tumor-draininglymph nodes were dispersed into PBS through a 40-nm cell strainer usingthe back of a 1-mL syringe plunger. All cell suspensions were washedonce with PBS, and the cell pellet was resuspended in 2% inactivatedfetal calf serum containing fluorophore-conjugated antibodies. Cellswere incubated for 15 minutes at 4° C. then fixed, permeabilized, andstained using a BioLegend FoxP3 staining kit. Samples were analyzed on aBD Fortessa flow cytometer. Antibodies (BioLegend) used in melanomaexperiments were: CD45-BV711 (clone 30-F11), CD8-BV650 (53-6.7),CD4-BV421 (GK1.5), TCRβ-BV510 (H57-597), CD25-AF488 (PC61), FoxP3-PE(MF-14). Antibodies (eBioscience) used in colon carcinoma experimentswere: CD45-BV510 (30-F11), CD3-BV 711 (17A2), CD49b-FITC (DX5),CD4-BV605 (GK1.5), CD8-PECy7 (53-6.7), Foxp3-APC (FJK-16s). FixableViability Dye eFluor 780 (eBioscience) was used to exclude dead cells.

Generation of Anti-Neoleukin-2/15 Polyclonal Antibody:

Mice were injected i.p. with 500 μg of K.O. neoleukin in 200 μL of a 1:1emulsion of PBS and Complete Freund's Adjuvant. Mice were boosted ondays 7 and 15 with 500 μg of K.O. neoleukin in 200 μL of a 1:1 emulsionof PBS and Incomplete Freund's Adjuvant. On day 20, serum was collectedand recognition of neoleukin-2/15 was confirmed by ELISA.

Enzyme-Linked Immunosorbent Assay (ELBA):

High-binding 96-well plates (Corning) were coated overnight at 4° C.with 100 ng/mL of neoleukin-2/15, mIL-2 (Peprotech), hIL-2 (Peprotech),or ovalbumin (Sigma-Aldrich) in carbonate buffer. Antibody binding totarget proteins was detected using HRP-conjugated sheep anti-mouse IgG(GE Healthcare) at 75 ng/mL. Plates were developed withtetramethylbenzidine and HCl. Absorbance was measured at 450 nm with anEnVision Multimode Plate Reader (PerkinElmer).

T Cell Proliferation Assay:

Cells were isolated from a mouse spleen using an Easy Sep T CellIsolation Kit (Stemcell Technologies). They were plated in RPMI in96-well culture plates at a density of 10,000 cells/well. Media weresupplemented with regular or heat-treated neoleukin-2/15, rmIL-2, orSuper-2. After 5 days of incubation at 37° C. cell survival andproliferation were measured by CellTiter-Glo Luminescent Cell ViabilityAssay (Promega).

Statistical and Power Analyses:

In vivo murine airway inflammation experiments: MIKEL. In vivo murineColon cancer experiments: CARLOS. In vivo murine Melanoma experiments:Comparisons of the survival of tumor-bearing mice were performed usingthe log-rank (Mantel-Cox) test. Comparisons of weight loss intumor-bearing mice were performed using a two-tailed t test. A P valueless than 0.05 was considered to be significant. The minimum group sizewas determined using G*Power for an expected large effect size (Cohen'sd=1.75).

Biolayer Interferometry analysis of a Mouse Serum Albumin (MSA) fusionto Neoleukin-2/15. Genetic fusion of Neoleukin-2/15 to MSA for extendedhalf-life and preserves intact binding affinity of the cytokine mimeticto murine IL-2RBeta and IL-2RGamma (33.5±0.2 nM) (data not shown). Theconstruct utilized in this study was as follows:

Optional: (HisTag TEV cleavage site  in parentheses)Mouse serum albumin (italicized) Linker Neo2/15 (bold font)(SEQ ID NO: 244) (GSDGGSHHHHHHGSGSENLYFQGSG) EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNIRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRORMIKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKINCDLYEKLGEYGFONAILVRYTOKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKD ALA GGGSGGSGGGSGGSGSGPKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS

Biotin-mIL2Gamma was immobilized on a Streptavidin biosensor, MSA-Neo2concentration was titrated from 729 to 1 nM in presence of saturatingconcentrations of mIL2Beta. Biolayer interferometry was carried out asabove: binding data were collected in a Octet RED96 (ForteBio, MenloPark, Calif.) and processed using the instrument's integrated softwareusing a 1:1 binding model. Biotinylated target receptors, either humanor murine IL-2Rα, IL-2Rβ, IL-2Rν, or human IL-4Rα, were functionalizedto streptavidin coated biosensors (SA ForteBio) at 1 μg/ml in bindingbuffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactantP20, 0.5% non-fat dry milk) for 300 seconds. Analyte proteins werediluted from concentrated stocks into binding buffer. After baselinemeasurement in binding buffer alone, the binding kinetics were monitoredby dipping the biosensors in wells containing 100 nM of the designedprotein (association) and then dipping the sensors back into baselinewells (dissociation).

CAR-T Cell In Vivo Experiments:

In vitro T cell proliferation assay. Primary human T cells were obtainedfrom healthy donors. Peripheral blood mononuclear cells (PBMC) wereisolated by centrifugation over Ficoll-Hypaque (Sigma). T cells wereisolated using EasySep™ CD8 or CD4 negative isolation kits (STEMCELLTechnologies). To stimulate T cells, T cells were thawed and incubatedwith anti-CD3/CD28 Dynabeads (Gibco) at 1:1 ratio in media supplementedwith 50 IU/ml (3.1 ng/ml) of IL2. Beads were removed after four days ofincubation. Stimulated or freshly thawed unstimulated T cells wereplated at 30000 or 50000 cells/well, respectively, in 96 well format andcultured in indicated concentrations of IL2 or neoleukin-2/15 intriplicate. Three days later, proliferation was measured usingCellTiter-Glo 2.0. (Promega).

In vivo RAJI experiment: Six- to eight-week old NSG mice were obtainedfrom the Jackson Laboratory. 0.5*10{circumflex over ( )}6 RAJI tumorcells transduced with ffluc/eGFP were tail vein injected into the NSGmice. Seven days post tumor inject, lentiviral transduced anti-CD19 CARTcells (0.4*10{circumflex over ( )}6 CD4, 0.4*10{circumflex over ( )}6CD8) prepared as described in (Liu et al, 2016) were infused i.v. intomice. hIL2 or neoleukin-2/15 at 20 μg/mouse were given i.p. from day 8to 16 post tumor injection.

Preparation of PEGylated Polypeptides:

Neo-2/15 stocks with either single or dual cysteine mutations weredialyzed into phosphate buffer, pH7.0 and adjusted to 1.0-2.0 mg/ml.TCEP was added at a molar ratio of 10:1 to protein and incubated for 10minutes at RT to reduce disulfides. Maleimide-modified PEG40k(PEG40k-MA) or PEG30k (PEG30k-MA) powder was added directly to thereduced protein solution at a molar ratio of 10:1 PEG:cysteine andincubated for 2 hours with stirring. Aliquots for SDS-PAGE were takendirectly from the reaction mixture. These data demonstrate the rapid,spontaneous, and near-quantitative formation of covalent linkagesbetween PEG40k-MA or PEG30k-MA and Neo-2/15 cysteine mutants in theexpected stoichiometry.

Treatment with Neo-2/15 and PEGylated Neo-2/15-E62C (Neo-2/15-PEG)Demonstrated Changes in the Levels of Multiple Inflammatory Markers:

Two non-human primates (NHP), one male and one female per group, wereassigned to treatment with either vehicle (group 1), Neo-2/15 (w/o PEG)(groups 2-4) or Neo-2/15 PEG (groups 5-7; single cysteine mutation ofE62C and PEG40K). Animals treated with vehicle or Neo-2/15 (w/o PEG)were dosed by intravenous (IV) bolus on study days 1, 2, 3, 4, 5, 6 and7 (once daily for one week) at dose levels of either 0 (vehicle) or doseadjusted values of 0.07, 0.21 or 0.14 mg/kg/day Neo 2/15 (w/o PEG)(groups 2, 3 and 4, respectively). Animals treated with Neo-2/15 PEGwere dosed by IV bolus on study days 1 and 7 at dose levels of 0.05,0.15 or 0.10 mg/kg/day Neo-2/15PEG (groups 5, 6 and 7, respectively).Cytokine samples were taken on day 1 and 7 at timepoints of 0, 4, 8 and24 hours post dose. Cytokine serum samples were prepared and frozen at<−70° C. and shipped for analysis where samples were analyzed through aLuminex multiplex immunoassays system. Several cytokines, includingIL-15 and IL-10 demonstrated marked differences in the time-course ofcytokine production, consistent with a more sustained pharmacodynamiceffect for the PEGylated molecule.

Targeted Neo-2/15 Fusions Retained their IL-2R Binding Affinity andDemonstrated Anti-Tumor Effects.

Select targeting domains were fused to the N- or C-termini of Neo-2/15via peptide linkers and were tested in vitro to characterize theirbinding affinity to human and mouse IL-2R by Biolayer Interferometry.The results confirmed that fusions to Neo-2/15 at either the N or Ctermini did not hinder its ability to bind IL-2R. Subsequent in vitroFlow Cytometry studies confirmed that the fusion proteins were capableof binding a target receptor on the surface of a cell. The efficacy ofthe targeted constructs was evaluated in in vivo mouse experiments, inwhich it was demonstrated that a targeted Neo-2/15 moiety to tumor cellsor immune cells has a beneficial anti-tumor effect over a non-targetedcontrol (data not shown).

Fusions that were tested include but are not limited to: (i) a fusion ofan anti-CD47 nanobody to the C terminus of Neo 2/15 via the linker ofSEQ ID NO:100; (b) a fusion of an anti-CD47 nanobody to the N terminusof Neo 2/15 via the linker of SEQ ID NO:100; (c) a fusion of ananti-CTLA4 nanobody to the C terminus of Neo 2/15 via the linker of SEQID NO:100; (d) a fusion of anti-CTLA4 nanobody to the N terminus of Neo2/15 via the linker of SEQ ID NO:100; (e) a fusion of an anti-PDL-1nanobody to the C terminus of Neo 2/15 via the linker of SEQ ID NO:100;and (f) a fusion of an anti-PDL-1 nanobody to the N terminus of Neo 2/15via the linker of SEQ ID NO:100.

Fusions of Albumin to Neo-2/15 Maintained IL-2R Binding Affinity.

Mouse serum albumin (MSA) was fused to the N-terminus of Neo 2/15 via apeptide linker and was tested in vitro to characterize its bindingaffinity to mouse IL-2R by Biolayer Interferometry. Biotin-mIL2Gamma wasimmobilized on a Streptavidin biosensor, MSA-Neo2 concentration wastitrated from 729 to 1 nM in presence of saturating concentrations ofmIL2Beta. The fusions maintained IL-2R binding capacity (data notshown).

PEGylated and Non-PEGylated Neo-2/15 does not Elicit a MeaningfulAnti-Drug Antibody (ADA) Response in Non-Human Primates (NHPs).

The potential of PEGylated and non-PEGylated Neo-2/15 (for PEGylatedNeo-2/15: single cysteine mutation of E62C and PEG40K) to elicit ADAswas tested in non-human primates. Animals were administeredintravenously with either compound for 1 week: PEGylated Neo-2/15 ondays 1 and 7; wild-type Neo-2/15 on days 1-7. Blood was drawn at varioustimes thereafter and analyzed for the presence of antibodies specificfor the administered compound. Each dose group consisted of 1 male and 1female macaque. Non-PEGylated Neo-2/15 was administered via daily ivbolus injection for 7 consecutive days at 0.1 m/kg, 0.2 mg/kg, or 0.3mg/kg. PEGylated Neo-2/15 was administered via iv bolus injection at0.015 mg/kg, 0.050 mg/kg, and 0.10 mg/kg on days 1 and 7. An equivalentvolume of saline was administered daily to a vehicle control group for 7consecutive days. Approximately 750 ul of blood was collected from eachanimal for ADA analysis on study Days 1 (pre-dose), 22, 29, and 43 viathe cephalic or saphenous vein. Serum was extracted from blood using aserum separator tube on wet ice and subsequently stored at −80C untilanalysis. All cynomolgus macaques receiving either vehicle or PEGylatedNeo-2/15 tested negative for ADAs on days 22, 29, and 43 demonstratingthat PEGylated Neo-2/15 did not elicit a detectable immune response,even after repeat dosing, despite being a computationally-designedprotein that is entirely foreign to the macaque immune system. Both (1male; 1 female) macaques receiving vehicle control tested negative forADAs against wild-type Neo-2/15 on days 1, 15, 22, and 28. All animals(3 males; 2 females) in the groups receiving non-PEGylated Neo-2/15tested negative for ADAs on day 1 (pre-dose). Of these, 3 out of 5 (60%)remained negative for ADAs on days 22, 29, and 43. The remaining twoanimals subsequently tested positive for ADAs on days 22, 29, or 43. Onesubject tested positive on days 22 and 29, but returned negative by day43. For that subject, the ADA response was low and transient, suggestingminimal clinical significance. Another subject tested positive on days22, 29, and 43. For that subject, the measured ADA concentrations werewell below 100 ng/ml and thus of unclear clinical relevance.

Data Tables

TABLE E1 Characterization of several de novo designed mimetics ofIL-2/IL-15. The table shows the Kd of de novo IL-2/IL-15 mimetics andreference cytokines for: mIL- 2Rβ, mIL-2Rβ 

 c, EC₅₀, the sequence similarity by structural alignment (MICAN ⁶³)against hIL-2 (PDB: 2B5I) and mIL-2 (PDB:), the parent of each molecule,its amino acid length, and the sequences for the de novo IL-2 mimetics.“N/S” stands for non-significant and “N/A” for non-available. Bindingaffinity (Kd) to HsIL-2Rβ 

 c, and cell signaling in human NK (YT, CD25-) cells EC50 Seq Seq Kd Kd(CD25-) identity to identity to HsIL- HsIL- pSTAT5p HsIL-2 MmIL-2 (%/2Rβ 

 c 2Rβ (nM)/ (%/(num (num Exp. Parent a.a. Name (nM) (nM) (exp i.d.)a.a. algn)) a.a. algn)) optimized molecule length HsiL-2 193.6 326.90.41/(a) 100.0/(120) 54.5/(112) — — 133 MmIL-2 8034.0 4950.0 39.05/(a)54.5/(112) 100/(122) — — 130 Super-2/ 300.9 2.0 0.07/(a) 949/(117)509/(114) Y HsIL-2 133 Superkine (PDB: 3QAZ) G1_ne02_40 260.0 1457.00.14/(b) 47.7/(86) 30.4/(79) N — 87 G1_ne02_41 187.0 720.6 0.07/(b)47.7/(86) 30.4/(79) N — 87 G1_ne02_43 533.4 2861.0 0.21/(b) 50.0/(86)32.9/(79) N — 87 G1_ne02_40_1F 2.3 2.6 0.09/(c) 44.2/(86) 26.6/(79) YG1_neo2_40 87 G2_ne02_40_1F_d 113.9 27.6 0.12/(a) 33.7/(89) 17.6/(85) NDe novo mimetic 100 sn36 design inspired on template: G1_ne02_40_1FNeoleukin-2/15 18.8 11.2 0.05/(a) 29.2/(89) 15.7/(83) Y (G2_ne02_40_ 100G2_neo2_40_1F 1F_dsn36 dsn36_opt) Binding affinity (Kd) to MmIL-2Rβ 

 c, and cell signaling (EC50) in murine T (CTLL-2, CD25+) cells EC50 SeqSeq Kd Kd (CD25+) identity to identity to MmIL- MmIL- pSTAT5 HsIL-2MmIL-2 2Rβ 

 c 2Rβ (nM)/ (%/(num (%/(num Exp. Parent a.a. Name (nM) (nM) (exp i.d.)a.a. algn)) a.a. algn)) optimized molecule length HsiL-2 492.2 8106.00.002/(d) *see top table MmIL-2 126.2 1496.0 0.003/(e) *see top tableSuper-2/ 312.2 214.0 N/A *see top table Superkine (PDB: 3QAZ)G1_ne02_40_1F 7.9 485.5 0.2/(e) *see top table G1_neo2_40_1F_H 2654.06799.0 37.38/(d) 39.5/(86) 25.0/(80) Y G1_neo2_40_1F 87 1G1_neo2_40_1F_H 963.7 68300.0 9.38/(d) 40.7/(86) 26.2/(80) YG1_neo2_40_1F 87 2 G1_neo2_40_1F_H 3828.0 N/S 35.2/(d) 39.5/(86)25.0/(80) Y G1_neo2_40_1F 87 3 G1_ne02_40_1F_H 391.8 10070.0 0.93/(d)41.9/(86) 26.2/(80) Y G1_neo2_40_1F 87 4 G1_neo2_40_1F_H 5123.0 45300.084.69/(d) 39.5/(86) 23.8/(80) Y G1_neo2_40_1F 87 5 G1_neo2_40_1F_ 4.3213.9 0.007/(d) 36.0/(86) 25.0/(80) Y G1_neo2_40_1F 87 M1 G1_neo2_40_1F_886.3 2599.0 3.11/(d) 37.2/(86) 25.0/(80) Y G1_ne02_40_1F 87 M-2G1_ne02_40_1F_ 64.8 402.3 0.08/(d) 34.9/(86) 25.3/(79) Y G1_neo2_40_1F87 M3 G2_neo2_40_1F_s 80.0 N/A 1.95/(f) 38.4/(86) 23.8/(80) N Sequence87 eq04 redesign of G1_ne02_40_1F G2_ne02_40_1F_s 39.1 N/A 1.74/(f)38.4/(86) 25.3/(79) N Sequence 87 eq12 redesign of G1_ne02_40_1FG2_neo2_40_1F_s 71.5 N/A 2.20/(f) 34.9/(86) 22.5/(80) N Sequence 87 eq16redesign of G1_ne02_40_1F G2_neo2_40_1F_s 27.8 N/A 1.06/(f) 39.5/(86)25.3/(79) N Sequence 87 eq26 redesign of G1_ne02_40_1F G2_neo2_40_1F_s13.6 N/A 0.24/(f) 36.0/(86) 25.0/(80) N Sequence 87 eq27 redesign ofG1_ne02_40_1F G2_neo2_40_1F_d 38.2 N/A 0.48/(f) 36.6/(82) 8.9/(90) N Denovo mimetic 107 sn29 design using template: G1_ne02_40_1FG2_neo2_40_1F_d 925.0 N/A 7.61/(f) 33.0/(97) 23.4/(94) N De novo mimetic107 sn30 design using template: G1_ne02_40_1F G2_neo2_40_1F_d 568.52432.0 1.36/(e) *see top table sn36 G2_neo2_40_1F_d 69.2 N/A 0.50/(f)33.7/(89) 17.9/(84) N De novo mimetic 100 sn40_ design inspired ontemplate: G1_ne02_40_1F Neoleukin-2/15 38.4 16.1 0.07/(e) *see top table(G2_ne02_40_1F_ dsn36_opt)

TABLE E2 Crystallographic data table for neoleukin-2/15 andneoleukin-2/15 quaternary complex with mIL-2Rβ

_(c). Neoleukin-2/15 Neoleukin-2/15 ternary complex (6DG6) with IL-2R(6DG5) Wave length 3928 − 1.999 47.005 − 2.516 Resolution range (2.07 −1.999) (2.828 − 2.516) Ellipsoidal resolution limit (Å) — 3.687 (0.065a* + 0.998 c*) (direction) — 3.756 (0.884 a* + 0.468 c*) — 2.516 (0.132a* + 0.859 b* + 0.495 c*) Space group P21 21 21 P 21 2 21 Unit cell (Å,°) 73.73, 86.8, 92.31, 90, 90, 90 65.125, 67.914, 172.084, 90, 90, 90Total reflections 351741 (32344) 132356 (7834) Unique reflections 40650(3977) 13961 (698) Multiplicity 8.7 (8.1) 9.5 (11.2) Completeness(spherical) (%) 92.58 (77.83) 52.3(9.0) Completeness (ellipsoidal) (%)93.2 (77.2) Mean I/sigma(I) 12.19 (1.25) 6.8 (1.3) Wilson B-factor 34.5439.86 R-merge 0.1027 (1.709) 0.359(2.516) R-meas 0.1094 (1.824) 0.380(2.636) R-pim 0.0369 (0.6252) 0.122 (0.780) CC1/2 0.999 (0.557) 0.987(0.445) CC* 1 (0.646) 0.993 (0.328) Resolution range used in refinement39.28 − 1.999 (2.07 − 1.999) 43.82 − 2.516 (2.606 − 2.516) Reflectionsused in refinement 37747 (3125) 13923 (135) Reflections used for R-free1840 (143) 1366 (14) R-work 0.2037 (0.3137) 0.2211 (0.3271) R-tree0.2260 (0.3377) 0.2658 (0.4429) Number of non-hydrogen atoms 4791 4100macromolecules 4735 3949 ligands — 138 solvent 56 13 Protein residues597 492 RMS(bonds) 0.005 0.004 RMS(angles) 0.88 0.94 Ramachandranfavored (%) 97.41 97.1 Ramachandran allowed (%) 2.59 2.9 Ramachandranoutliers (%) 0 0 Rotamor outliers (%) 1.26 4.5 Clashscore 2.14 4.55Average B-factor 52.56 47.05 macromolecules 52.54 46.39 ligands — 67.79solvent 54.21 27.31 Number of TLS groups 20 3 *Statistics for thehighest-resolution shell ate shown M parentheses.

TABLE S1 Amino acid sequences for the best twelvefirst-round designs. Ten of the designs were (G1_neo2_35-44) were experimentally  characterized by yeast display and all but two (G1_neo2_35and G1_neo2_44) were found to bind fluorescently labeledchimeric ILRβγ_(c) at low nanomolar  concentrations via flowcytometry screening of designed first-round protein binders.Designs indicated were expressed onyeast and incubated with 2 nM hIL-2Rβ

_(c) or 0 nM IL-2Rβ

_(c) (data not shown). Design Sequence G1_neo2_33STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDLDKAEDIRRNSDQARREAEKRGIDV RDLISNAQVILLEAR (SEQ ID NO: 103)G1_neo2_34 STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSCISTGKCDLDKAEDIRRNSDQARREAEKRGIDV RDLISNAQVILLEAR (SEQ ID NO: 104)G1_neo2_35 STKKWQLQAEHALLDWQMALNKSPEPNENLNRAITAAQSWISTGKIDCDKAEDIRRNSDQARREAEKRGIDV RDLISNAQVILLEAC (SEQ ID NO: 105)G1_neo2_36 STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELARNLEKVRDEALKRGIDV RDLVSNAKVIALELK (SEQ ID NO: 106)G1_neo2_37 STKKLQLQAEHFLLDVQMILNESPEPNEELNRCITDAQSWISTGKIDLDRAEECARNLEKVRDEALKRGIDV RDLVSNAKVIALELK (SEQ ID NO: 107)G1_neo2_38 STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSCISTGKCDLDRAEELARNLEKVRDEALKRGIDV RDLVSNAKVIALELK (SEQ ID NO: 108)G1_neo2_39 STKKLQLQAEHFLLDVQMILNESPEPNEELNRAITDAQSWISTGKIDLDRAEELCRNLEKVRDEALKRGIDV RDLVSNACVIALELK (SEQ ID NO: 109)G1_neo2_40 STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSWISTGKIDLDGAKELAKEVEELRQEAEKRGIDV RDLASNLKVILLELA (SEQ ID NO: 110)G1_neo2_41 STKKLQLQAEHALLDAQMMLNRSPEPNEKLNRIITTMQSCISTGKCDLDGAKELAKEVEELRQEAEKRGIDV RDLASNLKVILLELA (SEQ ID NO: 111)G1_neo2_42 STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMAKEAEKIRKEMEKRGIDV RDLISNIIVILLELS (SEQ ID NO: 112)G1_neo2_43 STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSCISTGKCDLDNAQEMAKEAEKIRKEMEKRGIDV RDLISNIIVILLELS (SEQ ID NO: 113)G1_neo2_44 STKKIQLQLEHALLDVQMALNRSPEPNESLNRMITWLQSWISTGKIDLDNAQEMCKEAEKIRKEMEKRGIDV RDLISNICVILLELS (SEQ ID NO: 114)

TABLE S2 Amino acid sequences    for the experimentallyoptimized first-round designs. Design Sequence G1_neo2_STKKTQLLAEHALLDAFMMLNVVPEPNEKLNRIITTM 40_1AQSWIYIGKIDADGAKELAKEVEELEQEYEKRGIDVED DASNLKVILLELA (SEQ ID NO: 115)G1_neo2_ STKKTQLLAEHALLDAHMMLNMLPEPNEKLNRIITTM 40_1BQSWIHTGKIDGDGAQELAKEVEELEQEYEKRGIDVED EASNLKVILLELA (SEQ ID NO: 116)G1_neo2_ STKKTQLLAEHALLDAFMMLNMVPEPNEKLNRIITTM 40_1CQSWIFTGKIDGDGAKELAKEVEELEQEFEKRGIDVED EASNLKVILLELA (SEQ ID NO: 117)G1_neo2_ STKKTQLLAEHALLDALMMLNMVPEPNEKLNRIITTM 40_1DQSWIFTGKIDGDGAQELAKEVEELEQELEKRGIDVED YASNLKVILLELA (SEQ ID NO: 118)G1_neo2_ STKKTQLLAEHALLDAHMMLNVVPEPNEKLNRIITTM 40_1EQSWITTGKIDRDGAQELAKEVEELEQELEKRGIDVDD DASNLKVILLELA (SEQ ID NO: 119)G1_neo2_ STKKTQLLAEHALLDALMMLNLLPEPNEKLNRIITTM 40_1FQSWIFTGKIDGDGAQELAKEVEELEQEHEKRGIDVE DYASNLKVILLELA (SEQ ID NO: 120)G1_neo2_ STKKTQLLAEHALLDAYMMLNMVPEPNEKLNRIITTM 40_1GQSWILIGKIDSDGAQELAKEVEELEQELEKRGIDVDD DASNLKVILLELA (SEQ ID NO: 121)G1_neo2_ STKKTHLLAEHALLDAYMMLNVMPEPNEKLNRIITTM 40_1HQSWIFTGKIDGDGAKELAKEVEELEQEFEKRGIDVDD DASNLKVILLELA (SEQ ID NO: 122)G1_neo2_ STKKTQLLAEHALLDAYMMLNLVPEPNEKLNRIITTM 40_1IQSWIFTGKIDADGAQELAIEVEELEQEYEKRGIDVDD YASNLKVILLELA (SEQ ID NO: 123)G1_neo2_ STKKTQLMAEHALLDAFMMLNVLPEPNEKLNRIITTM 40_1JQSWIFTGKIDGDDAQELAKEVEELEQELEKRGIDVDD DASNLKVILLELA (SEQ ID NO: 124)G1_neo2_ STKKTQLLIEHALLDALDMSRNLPEPNEKLSRIITTM 40_1F_H1QSWIFTGKIDGDGAQQLAKEVEELEQEHEKRGEDVED EASNLKVILLELA (SEQ ID NO: 125)G1_neo2_ STKKTQLLLEHALLDALHMRRNLPEPNEKLSRIITTM 40_1F_H2QSWIFTGKIDGDGAQELAKEVEELEQEHEKRGRDVED   DASNLKVILLELA (SEQ ID NO: 126)G1_neo2_ STKKTQLLIEHALLDALNMRKKLPEPNEKLSRIITDM 40_1F_H3QSWIFTGKIDGDGAQQLAKEVEELEQEHEKRGGDVED YASNLKVILLELA (SEQ ID NO: 127)G1_neo2_ STKKTQLLLEHALLDALHMSRELPEPNEKLNRIITDM 40_1F_H4QSWIFTGKIDGDGAQDLAKEVEELEQEHEKRGGDVED YASNLKVILLELA (SEQ ID NO: 128)G1_neo2_ STKKTQLLIEHALLDALHMSRKLPEPNEKLSRIITTM 40_1F_H5QSWIFTGKIDGDGAQHLAKEVEELEQEHEKRGGEVED EASNLKVILLELA (SEQ ID NO: 129)G1_neo2_ STKKTQLLIEHALLDALHMKRKLPEPNEKLNRIITNM 40_1F_H6QSWIFTEKIDGDGAQDLAKEVEELEQEHEKRGQDVED YASNLKVILLELA (SEQ ID NO: 130)G1_neo2_ STEKTQLAAEHALRDALMLKHLLNEPNEKLARIITTM 40_1F_M1QSWQFTGKIDGDGAQELAKEVEELQQEHEVRGIDVED YASNLKVILLHLA (SEQ ID NO: 131)G1_neo2_ STKNTQLAAEDALLDALMLRNLLNEPNEKLARIITTM 40_1F_M2QSWQFTEKIDGDGAQELAKEVEELQQEHEERGIDVED   YASNLKVILLQLA (SEQ ID NO: 132)G1_neo2_ STEKTQHAAEDALRDALMLRNLLNEPNEKLARIITTM 40_1F_M3QSWQFTEKIDGDGAQELAKEVEELQQEHEVRGIDVED YASNLKVILLQLA (SEQ ID NO: 133)

TABLE S3 Amino acid sequences for second-round designs.G2_neo2_40_1F_seq02 to G2_neo2_40_1F_seq28 correspond to the 27 Rosetta sequenceredesigns of G1_neo2_40_1F; G2_neo2_40_1F_ seq29 to G2_neo2_40_1F_seq42 representthe 14 new de novo mimetic designs. Design Sequence G2_neo2_40_1TQKKQQLLAEHALLDALMIINMLKTSSEAVNR F_seq02MITIAQSWIFTGTSNPEEAIKEMIKMAEQAEE EARREGVDTEDYVSNLKVILKEIA(SEQ ID NO: 134) G2_neo2_40_1 TTKKYQLLVEHALLDALMMLNLSSESNEKENR F_seq03IITTMQSWIFTGTFDPDQAEELAKVEELRE EFRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 135) G2_neo2_40_1 TKKIQLLVEEALLDALMILNLSSESNEKLNRI F_seq04ITTLQSWIFRGEIDPDRARELAKLLEEIREEM RKRGIDTEDYVSNMIVIIRELA (SEQ ID NO: 136) G2_neo2_40_1 TKKKIQLLAEHVLLDLLMMLNLSSESNEKMNR F_seq05LITIVQSWIFTGTIDPDQAEEMAKWVEELREE FRKRGIDTEDYASNVKVILKELS (SEQ ID NO: 137) G2_neo2_40_1 TKKKYQLLIEHLLLDALMVLNMSSESNEKLNR F_seq06IITILQSWIFTGTWDPDLAEEMEKLMQEIEEE LRRRPGIDTEDYMSNMRVIIKELS (SEQ ID NO: 138) G2_neo2_40_1 TKKKLQLLVEHLLLDMLMILNMSSESNEKLNR F_seq07LITELQSWIFRGEIDPDKAEEMWKIMEEIE KELRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 139) G2_neo2_40_1 TSKKQQLLAEHALLDALMILNISSESSEAVNR F_seq08AITWLQSWIFKGTVNPDQAEEMRKLAEQIREE MRKRGIDTEDYVSNLEVIAKELS (SEQ ID NO: 140) G2_neo2_40_1 TKKKYQLLIEHLLLDLLMVLNMSSESNEKINR F_seq09LITWLQSWIFTGTYDPDLAEEMYKILEELREE MRERGIDTEDYMSNMRVIVKELS (SEQ ID NO: 141) G2_neo2_40_1 TKKKWQLLIEHLLLDLLMILNLSSSSNEKLNR F_seq10LITWLQSWIFTGTYDPDLAEEMKKMMDEIED ELRERGIDTEDYMSNAKVIIKELS (SEQ ID NO: 142) G2_neo2_40_1 TKKKIQLLVEHALLDALMILNLSSESNSKLNR F_seq11IITTMQSWIFTGTIDPDQAEELSKLVEEIREE MRKRGIDTEDYVSNLKVILDELS (SEQ ID NO: 143) G2_neo2_40_1 TEKKLQLLVEHALLDALMILNLWSESNEKLNR F_seq12IITTMQSWIFTGRIDPDKAEELAKLVEELREE ARERGIDTEDYVSNLKVTLKELS (SEQ ID NO: 144) G2_neo2_40_1 TKKKYQLLMEHLLLDLLMVLNMSSESNEKLNR F_seq13LITIIQSWIFTGTWDPDKAEEMAKMLKEIEDE LRERGIDTEDYMSNMIVIMKELS (SEQ ID NO: 145) G2_neo2_40_1 TTKKIQLLVEHALLDALMLLNLSSESNEKMNR F_seq14IITTMQSWIFEGRIDPDQAQELAKLVEELREE FRKRGIDTEDYVSNLKVTLEELS (SEQ ID NO: 146) G2_neo2_40_1 TKKKIQLLVEHALLDAIMMLNLSSESNEKLNR F_seq15IITTMQSWIFTGTIDPDQAEELAKLVRELREE FRKRGIDTEDYASNLEVILRELS (SEQ ID NO: 147) G2_neo2_40_1 TKKKIQLLVEHALLDALMILNLSSKSNEKLNR F_seq16IITTMQSWIFNGTIDPDRARELAKLVEEIRDE MEKNGIDTEDYVSNLKVILSELA (SEQ ID NO: 148) G2_neo2_40_1 TKKKYQLLIEHVLLDLLMLLNLSSESNEKMNR F_seq17LITILQSWIFTGTYDPDKAEEMAKLLKELREE FRERGIDTEDYISNAIVILKELS (SEQ ID NO: 149) G2_neo2_40_1 TKKKIQLLVEHALLDALMMLNLSSESNEKLNR F_seq18IITTMQSWIFTGTIDPDRAEELAKLVEELREE FRKRGIDTEDYASNLKVILKELS (SEQ ID NO: 150) G2_neo2_40_1 TKKKIQLLVEHALLDALMMLNLSSESNEKLNR F_seq19IITTMQSWIFNGTIDPDQARELAKLVEELREE FRKRGIDTEDYASNLKVILEELA (SEQ ID NO: 151) G2_neo2_40_1 TKKKLQLLVEHALLDALMLLNLSSESNEKLNR F_seq20IITTMQSWIFTGTVDPDQAEELAKLVEEIREE LRKRGIDTEDYVSNLKVTLKELS (SEQ ID NO: 152) G2_neo2_40_1 TTKKYQLLVEHALLDALMILNLSSESNEKLNR F_seq21IITTMQSWIFTGTFDPDQAEELAKLVREIREE MRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 153) G2_neo2_40_1 TKKKIQLLVEHALLDALMILNLSSESNEKLNR F_seq22IITTMQSWIFTGTIDPDRAEELAKLVREIREE MRKRGIDTEDYVSNLEVILRELS (SEQ ID NO: 154) G2_neo2_40_1 TKKKYQLLIEHLLLDLLMILNLSSESNEKLNR F_seq23LITWLQSWIFRGEWDPDKAEEWAKILKEIREE LRERGIDTEDYMSNAIVIMKELS(SEQ ID NO: 155) G2_neo2_40_1 TDKKLQLLVEHLLLDLLMMLNLSSKSNEKMNR F_seq24LITIAQSWIFTGKVDPDLAREMIKLLEETEDE NRKNGIDTEDYVSNARVIAKELE (SEQ ID NO: 156) G2_neo2_40_1 TKKKIQLLVEHALLDALMLLNLSSESNEKMNR F_seq25IITTMQSWIFTGTIDPDQAEELAKLVEELKEE FKKRGIDTEDSYVSNLKVILKELS (SEQ ID NO: 157) G2_neo2_40_1 TKKYQLLIEHALLDALMILNLWSESNEKLNRI F_seq26ITTMQSWIFTGTYDPDKAEELEKLAKEIEDEA RERGIDTEDYMSNLRVILKSLS (SEQ ID NO: 158) G2_neo2_40_1 TKKKAQLLAEHALLDALMLLNLSSHESNERLN F_seq27RIITWLQSIIFTGTYDPDMVKEAVKLADEIED MRKRGIDTEDYVSNLRVILQELA (SEQ ID NO: 159) G2_neo2_40_1 TQKKNQLLAEHLLLDALMVLNQSSESSEVANR F_seq28IITWAQSWIFEGRVDPNKAEEAKKLAKKLEEE MRKRGIDMEDYISNMKVIAEEMS (SEQ ID NO: 160) G2_neo2_40_1 EDYYSNLKVILEELAREMERNGLSDKAEEWRQ F_seq29WKKIVERIRQIRSNNSDLNEAKELLNRLITYI QSQIFEISERIRETDQEKKEESWKKWQLLLEHALLDVLMLLND (SEQ ID NO: 161) G2_neo2_40_1PEKKRQLLLEHILLDALMLLNLLETNPQNTES F_seq30KFEDYISNAEVIAEELAKLMESLGLSDEAEKF KKIKQWLREVWRIWSSTNWSTLEDKARELLNRIITTIQSQIFY (SEQ ID NO: 162) G2_neo2_40_1PEKKRQLLLEHILLDLLMILNMIETNRENTES F_seq31EMEDYWSNVRVILRELARLMEELNYKELSELM ERMRKIVEKIRQIVTNNSSLDTAREWLNRLITWIQSLIFR (SEQ ID NO: 163) G2_neo2_40_1 PEKKRQLLAEHALLDALMLLNIIETNSKNTESF_seq32 KMEDYVSNLSVILTEFKKLAEKLNFSEEAERAERMKRWARKAYQMMTLDLSLDKAKEMLNRIIT ILQSIIFN (SEQ ID NO: 164) G2_neo2_40_1PEKKRQLLAEHLLLDVLMMLNGNASLKDYASN F_seq33AQVIADEFRELARELGLTDEAKKAEKIIEALE RAREWLLNNKDKEKAKEALNRAITIAQSWIFN (SEQ ID NO: 165) G2_neo2_40_1 PEKKRQLLLEHLLLDLLMILNMLRTNPKNIES F_seq34DWEDYMSNIEVIIEELRKIMESLGRSEKAKEW KRMKQWVRRILEIVKNNSDLEEAKEWLNRLITIVQSEIFS (SEQ ID NO: 166) G2_neo2_40_1 WEKKRQLLLEHLLLDLLMILNMWRTNPQNTESF_seq35 LMEDYMSNAKVIVEELARMMRSQGLEDKAREWEEMKKRIEEIRQIIQNNSSKERAKEELNRLIT YVQSEIFR (SEQ ID NO: 167) G2_neo2_40_1PKKKIQLLAEHALLDALMILNIVKTNSQNAEE F_seq36KLEDYASNVEVILEEIARLMESGDQKDEAEKA KRMKEWMKRIKTTASEDEQEEMANRIITLLQSWIFS (SEQ ID NO: 168) G2_neo2_40_1 PEKKRQLLAEHALLDALMILNILQTNPQNAEEF_seq37 KLEDYMSNVEVIMEEFARMMRSGDRSEEAENAERIKKWVRKASSTASSEEQREMMNRAITLMQS WIFE (SEQ ID NO: 169) G2_neo2_40_1PEKKRQLLAEHLLLDALMVLNMLTTNSKNTEE F_seq38KLEDYISNMKVIIKEMIELMRSLGRLEEAEKW KEALKAVEKIGSRMDSSTARELANRIITLAQSAIFY (SEQ ID NO: 170) G2_neo2_40_1 PEKKRQLLAEHALLDALMFLNLVETNPDQAEEF_seq39 KIEDYASNLRVIAEELARLFENLGRLDEAQKAKDIKELAERARSRVSSEKRKEAMNRAITILQS MIFR (SEQ ID NO: 171) G2_neo2_40_1PEKKRQLLAEHALLDALMILNIIRTMSDNTES F_seq40KLEDYISNLKVILEEIARLMESLGLSDEAEKA KEAMRLADKAGSTASEEEKKEAMNRVITWAQSWIFN (SEQ ID NO: 172) G2_neo2_40_1 PEKKRQLLAEHALLDALMMLNILRTNPDNAEEF_seq41 KLEDYWSNLIVILREIAKLMESLGLTDEAEKAKEAARWAEEARTTASKDQRRELANRIITLLQS WIFS (SEQ ID NO: 173) G2_neo2_40_1PEKKRQLLAEHLLLDALMILNIIETNEQNAES F_seq42KLEDYISNAKVILDEFREMARDLGLLDEAKKA EKMKRWLEKMRSNASSDERREWANRMITTAQSWIFN (SEQ ID NO: 174)

TABLE S4 Amino acid sequences for  the experimentallyoptimized second-round designs. Design Sequence G2_neo2_40_TNKEAQLHAEFALYDALMLLNLSSESNERLNRIITW 1F_seq27_S18LQSIIFYETYDPDMVKEAVKLADEIEDEMRKRKIDT EDYVVNLRLILQELA (SEQ ID NO: 175)G2_neo2_40_ TKKDAELLAEFALYDALMLLNLSSESNERLNEIITW 1F_seq27_S22LQSIIFYGTYDPDMVKEAVKLADEIEDEMRKRGIDT EDYVSNLRLILQELA (SEQ ID NO: 176)G2_neo2_40_ TNKKAQLHAEFALYDALMLLNLSSESNERLNDIITW 1F_seq27_S24LQSIIFTGTYDPDMVKEAVKLADEIEDEMRKRKIDT EDYVVNLRYILQELA (SEQ ID NO: 177)G2_neo2_40_ EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKI 1F_seq29_S6VERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEVLHGVGETDQEKKEESWKKWDLLLEHALLDVLMLLND  (SEQ ID NO: 178) G2_neo2_40_EDYYSNLKVILEELAREMERNGLSDKAEEWRQWKKI 1F_seq29_S7VERIRQIRSNNSDLNEAKELLNELITYIQSQIFEVIEREGETDQEKKEESWKKWELHLEHALLDVLMLLND  (SEQ ID NO: 179) G2_neo2_40_EDYYSNLKLILEELAREMERNGLSDKAEEWRQWKKIVERIRQIRSNNSDLNEAKELLNRLITYIQSQIFEVL 1F_seq29_S8EGVGETDQEKKEESWKKWELHLEHALLDVLMLLND  (SEQ ID NO: 180) Neolukin-2/15PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLED (i.e. G2_YAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMK neo2_40_1F_RIKTTASEDEQEEMANAIITILQSWIFS seq36_S11) (SEQ ID NO: 181) G2_neo2_40_PKKKIQLLAEHALFDLLMILNIVKTNSQNAEEKLED 1F_seq36_S12YAYNAGVILEEIARLFESGDQKDEAEKAKRMKEWMK RIKDTASEDEQEEMANEIITILQSWNFS (SEQ ID NO: 182)

Neoleukin-2/15-H8Y-K33E: H1->H3->H2′->H4 (SEQ ID NO: 94)PKKKIQLYAEHALYDALMILNIVKTNSPPAEEELEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIIT ILQSWIFS

Binding of Neoleukin-2/15-H8Y-K33E to the IL2 receptor was measured bybiolayer interferometry, and it was found to have higher bindingaffinity than Neoleukin-2 for IL2-Rbeta, both when tested againstIL2Rbeta alone and when tested against the IL2Rbeta-gamma complex. Thisincreased affinity was attributable mostly to an improved off-rate fromIL2-Rbeta.

TABLE S51 Amino acid sequences for theinterleukin-4 mimetic designs based on reengineering of neolukin-2/15.Design Sequence IL4_G2_neo2_40_1 PKKKIQITAEEALKDALSILNIVKINSPPAEF_seq36_S11 EQLERFAKRFERNLWGIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITI LQSWIFS (SEQ ID NO: 183) Neoleukin-4PKKKIQIMAEEALKDALSILNIVKINSPPAE (i.e. IL4_G2_EQLERFAKRFERNLWGIARLFESGDQKDEAE neo2_40_1KAKRMIEWMKRIKTTASEDEQEEMANAIITI F_seq36_S11_MIF)LQSWFFS (SEQ ID NO: 184)

1-138. (canceled)
 139. A non-naturally occurring polypeptide comprisingdomains X1, X2, X3, and X4, wherein: (a) X1 is a peptide comprising theamino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical toEHALYDAL (SEQ ID NO:1); (b) X2 is a helical-peptide of at least 8 aminoacids in length; (c) X3 is a peptide comprising the amino acid sequenceat least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 100% identical to YAFNFELI (SEQ ID NO:2);(d) X4 is a peptide comprising the amino acid sequence at least 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to ITILQSWIF (SEQ ID NO:3); wherein X1, X2,X3, and X4 may be in any order in the polypeptide; wherein amino acidlinkers may be present between any of the domains; and wherein thepolypeptide binds to IL-2 receptor βν_(c) heterodimer (IL-2Rβν_(c)),IL-4 receptor αν_(c) heterodimer (IL-4Rαν_(c)), or IL-13 receptor αsubunit (IL-13Rα).
 140. The polypeptide of claim 139, wherein: (i) X1includes one or both of the following: H at residue 2 and Y at residue5; and/or (ii) X3 includes 1, 2, 3, 4, or all 5 of the following: Y atresidue 1, F at residue 3, N at residue 4, L at residue 7, and I atresidue 8; and/or. (iii) X4 includes I at residue
 8. 141. Thepolypeptide of claim 139, wherein: (i) X1 includes E at residue 2 and Kat residue 5; and (ii) X3 includes F at residue 1, K at residue 3, R atresidue 4, R at residue 7, and N at residue
 8. 142. The polypeptide ofclaim 141, wherein (iii) X4 includes F at residue
 8. 143. Thepolypeptide of claim 139, wherein: X1 is a peptide comprising the aminoacid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4); X3 is a peptide comprising theamino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to thepeptide LEDYAFNFELILEEIARLFESG (SEQ ID NO:5); and X4 is a peptidecomprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6).
 144. Thepolypeptide of claim 143, wherein: (i) X1 includes 1, 2, 3, 4, or all 5of the following: L at residue 7, H at residue 8, H at residue 11, Y atresidue 14; Mat residue 18; and/or (ii) X3 includes 1, 2, 3, 4, 5, 6, 7,or all 8 of the following: D at residue 3, Y at residue 4, F at residue6, N at residue 7, L at residue 10, I at residue 11, E at residue 13, orE at residue 14; and/or. (iii) X4 includes I at residue
 19. 145. Thepolypeptide of claim 139, wherein: X1 is a peptide comprising the aminoacid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the peptidePKKKIQIMAEEALKDALSILNI (SEQ ID NO: 8); X3 is a peptide comprising theamino acid sequence at least 37% 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 100% identical to the peptideLERFAKRFERNLWGIARLFESG (SEQ ID NO: 9); and X4 is a peptide comprisingthe amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to thepeptide EDEQEEMANAIITILQSWFFS (SEQ ID NO: 10). wherein (i) X1 includes Iat residue 7, T or M at residue 8, E at residue 11, K at residue 14 andS at residue 18; and (ii) X3 includes R at residue 3, F at residue 4, Kat residue 6, R at residue 7, R at residue 10, N at residue 11, W atresidue 13, and G at residue
 14. 146. The polypeptide of claim 145,wherein (iii) X4 includes F at residue
 19. 147. The polypeptide of claim139, wherein: (i) X1 includes: E at residue 1, L at residue 4, Y atresidue 5, D at residue 6 and L at residue 8; (ii) X3 includes: Y atresidue 1, N at residue 4, L at residue 7, and I at residue 8; and (iii)X4 includes: I at residue 1, Q at residue 5, and W at residue
 7. 148.The polypeptide of claim 143, wherein: (i) X1 includes: E at residue 10,L at residue 13, Y at residue 14, D at residue 15 and L at residue 17;(ii) X3 includes: L at residue 1, Y at residue 4, N at residue 7, L atresidue 10, I at residue 11; and I at residue 15; and (iii) X4 includes:I at residue 12, Q at residue 16, and W at residue
 18. 149. Thepolypeptide of claim 145, wherein: (i) X1 includes: E at residue 10, Eat residue 11, A at residue 12, L at residue 13, K at residue 14, D atresidue 15, A at residue 16, and L at residue 17; (ii) X3 includes: F atresidue 4, A at residue 5, K at residue 6, R at residue 7, F at residue8, E at residue 9, R at residue 10, and N at residue 11; and (iii) X4includes: I at residue 11, I at residue 12, T at residue 13, I atresidue 14, L at residue 15, Q at residue 16, S at residue 17, W atresidue 18, F at residue 19, F at residue
 20. 150. The polypeptide ofclaim 143, wherein: (i) X1 is a peptide comprising an amino acidsequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the peptidePKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4), wherein the amino acid atposition 1 is P or if substituted is A, F, I, L, M, Q, R, S, or W; theamino acid at position 2 is K or if substituted is A, D, E, G, or V; theamino acid at position 3 is K or if substituted is D, E, F, or W; theamino acid at position 4 is K or if substituted is D, E, N, P, R, or W;the amino acid at position 5 is I or if substituted is D, E, H, K, L, M,or S; the amino acid at position 6 is Q or if substituted is A, D, E, G,L, P, S, or W; the amino acid at position 7 is L or if substituted is D,E, Q, Y, or I; the amino acid at position 8 is H or if substituted is A,F, W, Y, M, or T; the amino acid at position 9 is A or if substituted isC, F, or P; the amino acid at position 10 is E or if substituted is C,D, F, K, or P; the amino acid at position 11 is H or if substituted isD, F, or E; the amino acid at position 12 is A or if substituted is D,E, P, S, T, or V; the amino acid at position 13 is L or if substitutedis H, I, M, P, R, V, or W; the amino acid at position 14 is Y or ifsubstituted is F, R, W, or K; the amino acid at position 15 is D or ifsubstituted is E, N, or Y; the amino acid at position 16 is A or ifsubstituted is C, L, M, or S; the amino acid at position 17 is L or ifsubstituted is F, I, M, P, or R; the amino acid at position 18 is M orif substituted is G, Q, Y, or S; the amino acid at position 19 is I orif substituted is L, M, P, Q, or V; the amino acid at position 20 is Lor if substituted is A, K, M, Q, R, or S; the amino acid at position 21is N or if substituted is G, K, P, R, S, or W; the amino acid atposition 22 is I or if substituted is D, E, K, M, N, W, or Y; and (ii)X3 is a peptide comprising an amino acid sequence at least 25%, 27%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 100% identical to the peptide LEDYAFNFELILEEIARLFESG (SEQ IDNO:5), wherein: the amino acid at position 1 is L or if substituted isA; the amino acid at position 2 is E or if substituted is D, G, K, M, orT; the amino acid at position 3 is D or if substituted is E, N, Y, or R;the amino acid at position 4 is Y or if substituted is C, D, G, T, or F;the amino acid at position 5 is A or if substituted is F, H, S, V, W, orY; the amino acid at position 6 is F or if substituted is A, I, M, T, V,Y, or K; the amino acid at position 7 is N or if substituted is D, K, S,T, or R; the amino acid at position 8 is F or if substituted is A, C, G,L, M, S, or V; the amino acid at position 9 is E or if substituted is C,H, K, L, R, S, T, or V; the amino acid at position 10 is L or ifsubstituted is F, I, M, Y, or R; the amino acid at position 11 is I orif substituted is L, N, T, or Y; the amino acid at position 12 is L orif substituted is F, K, M, S, or V; the amino acid at position 13 is Eor if substituted is A, D, F, G, I, N, P, Q, S, T, or W; the amino acidat position 14 is E or if substituted is A, F, G, H, S, or V; the aminoacid at position 15 is I or if substituted is C, L, M, V, or W; theamino acid at position 16 is A or if substituted is D, G, S, T, or V;the amino acid at position 17 is R or if substituted is H, K, L, or N;the amino acid at position 18 is L or if substituted is C, D, G, I, Q,R, T, or W; the amino acid at position 19 is F or if substituted is D,M, N, or W; the amino acid at position 20 is E or if substituted is A,C, F, G, M, S, or Y; the amino acid at position 21 is S or ifsubstituted is D, E, G, H, L, M, R, T, V, or W; the amino acid atposition 22 is G or if substituted is A, D, K, N, S, or Y; and (iii) X4is a peptide comprising an amino acid sequence at least 25%, 27%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or100% identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6) theamino acid at position 1 is E or if substituted is D, G, K, or V; theamino acid at position 2 is D or if substituted is I, M, or S; the aminoacid at position 3 is E or if substituted is G, H, or K; the amino acidat position 4 is Q or if substituted is E, G, I, K, R, or S; the aminoacid at position 5 is E or if substituted is A, D, G, H, S, or V; theamino acid at position 6 is E or if substituted is C, D, G, I, M, Q, R,T, or V; the amino acid at position 7 is M or if substituted is C, E, L,P, R, or T; the amino acid at position 8 is A or if substituted is F, L,M, or W; the amino acid at position 9 is N or if substituted is A, G, L,Q, R, or T; the amino acid at position 10 is A or if substituted is C,D, E, F, H, I, or W; the amino acid at position 11 is I or ifsubstituted is M, N, S, V, or W; the amino acid at position 12 is I orif substituted is K, L, S, or V; the amino acid at position 13 is T orif substituted is C, L, M, R, or S; the amino acid at position 14 is Ior if substituted is L, P, T, or Y; the amino acid at position 15 is Lor if substituted is F, G, I, M, N, or V; the amino acid at position 16is Q or if substituted is H, K, or R; the amino acid at position 17 is Sor if substituted is C, F, K, W, or Y; the amino acid at position 18 isW or if substituted is K, Q, or T; the amino acid at position 19 is I orif substituted is C, G, or N; the amino acid at position 20 is F or ifsubstituted is C, G, L, or Y; and the amino acid at position 21 is S orif substituted is A, F, G, H, or Y.
 151. The polypeptide of claim 143,wherein X2 is a peptide comprising the amino acid sequence at least 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical along its length to KDEAEKAKRMKEWMKRIKT (SEQID NO:7).
 152. The polypeptide of claim 151, wherein X2 is a peptidecomprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identicalto the peptide KDEAEKAKRMKEWMKRIKT (SEQ ID NO:7) wherein: the amino acidat position 1 is K or if substituted is A, H, L, M, R, S, or V; theamino acid at position 2 is D or if substituted is A, E, Q, R, S, T, V,W, or Y; the amino acid at position 3 is E or if substituted is C, G, K,L, N, Q, R, or W; the amino acid at position 4 is A or if substituted isF, G, N, S, T, V, or Y; the amino acid at position 5 is E or ifsubstituted is A, G, I, M, R, V, or C; the amino acid at position 6 is Kor if substituted is C, E, L, N, R, or V; the amino acid at position 7is A or if substituted is C, E, I, L, S, T, V, or W; the amino acid atposition 8 is K or if substituted is H, L, M, S, T, W, or Y; the aminoacid at position 9 is R or if substituted is A, I, L, M, Q, or S; theamino acid at position 10 is M or if substituted is A, I, S, W, or Y;the amino acid at position 11 is K or if substituted is C, I, L, S, orV; the amino acid at position 12 is E or if substituted is C, K, L, P,Q, R, or T; the amino acid at position 13 is W or if substituted is A,D, H, or N; the amino acid at position 14 is M or if substituted is A,C, G, I, L, S, T, or V; the amino acid at position 15 is K or ifsubstituted is A, E, G, I, L, M, R, or V; the amino acid at position 16is R or if substituted is G, H, L, S, T, V, or C; the amino acid atposition 17 is I or if substituted is A, L, or V; the amino acid atposition 18 is K or if substituted is A, C, D, E, G, H, I, M, or S; andthe amino acid at position is 19 is T or if substituted is D, E, G, L,N, or V.
 153. The polypeptide of claim 139, wherein the polypeptidecomprises a polypeptide at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to theamino acid sequence selected from the group consisting of SEQ IDNOS:11-94, 103-184, 190-243, and 245-247.
 154. The polypeptide of claim153 comprising the amino acid sequence set forth in SEQ ID NO:
 90. 155.The polypeptide of claim 153 comprising the amino acid sequence setforth in SEQ ID NO:
 181. 156. The polypeptide of claim 139, wherein thedomains are arranged N-terminal to C-terminal in an arrangement selectedfrom the group consisting of X1-X3-X2-X4.
 157. The polypeptide of claim139, wherein the polypeptide further comprises at least one disulfidebond.
 158. The polypeptide of claim 157 wherein the disulfide bond linkstwo of the X1, X2, X3, and X4 domains together.
 159. The polypeptide ofclaim 158 wherein the disulfide bond links the X1 domain to the X4domain.
 160. The polypeptide of claim 155, wherein the polypeptidefurther comprises at least one disulfide bond provided that theN-terminal proline and the C-terminal serine of SEQ ID NO:181 are absentand the amino acid sequence CNSN (SEQ ID NO:260) is present at theN-terminus of SEQ ID NO: 181 and the amino acid sequence NFQC (SEQ IDNO:261) is present at the C terminus of SEQ ID NO:181.
 161. Thepolypeptide of claim 139, wherein the polypeptide is linked to astabilization compound selected from polyethylene glycol or albumin.162. The polypeptide of claim 139, wherein the polypeptide furthercomprises a targeting domain.
 163. The polypeptide of claim 162, whereinthe targeting domain is a translational fusion with the polypeptide.164. The polypeptide of claim 163 wherein the targeting domain binds toa cell surface protein.
 165. The polypeptide of claim 163, wherein thetargeting domain binds to a tumor cell, tumor vascular component cell,tumor microenvironment cell surface marker, or an immune cell surfacemarker.
 166. The polypeptide of claim 162, wherein the targeting domainis an scFv, a F(ab), a F(ab′)₂, a B cell receptor (BCR), a DARPin, anaffibody, a monobody, a nanobody, diabody, an antibody (including amonospecific or bispecific antibody) or a cell-targeting oligopeptide.167. A crystalline form of a polypeptide that binds to the IL-2 receptorβν_(c) heterodimer (IL-2Rβν_(c)), having a three dimensional structurewith the structural coordinates of Table E2 and comprising the aminoacid sequence set forth in SEQ ID NO:
 181. 168. A pharmaceuticalcomposition, comprising the polypeptide of claim 139, and apharmaceutically acceptable carrier.